Neuroprotective peptides

ABSTRACT

The invention relates to the use of an isolated peptide of 10 to 32 amino acid residues in length for the treatment of neural injury, wherein the isolated peptide comprises at least 10 to 22 arginine residues. The peptide may be a poly-arg sequence or an arginine-rich peptide.

FIELD OF THE INVENTION

This invention relates to peptides having neuroprotective activity, thepeptides being useful in treating stroke and other neural injuries ordisorders. The invention relates further to a method of treating aneural injury or disorder using the peptides of the invention.

BACKGROUND OF THE INVENTION

Cell penetrating peptides (CPPs) are small peptides that are used tofacilitate the delivery of normally non-permeable cargos such as otherpeptides, proteins, nucleic acids or drugs into cells.

The development of cell penetrating peptides (CPPs), also referred to aspeptide transduction domains (PTDs), as facilitators of therapeutic drugdelivery has progressed significantly since the initial discovery of aPTD within the human immunodeficiency virus-type 1 trans-activatingtranscriptional activator (Frankel and Pabo, 1988; Green andLoewenstein, 1988), commonly referred to as TAT. Since then, the activetransporting portion of this sequence has been isolated (TAT₄₈₋₅₅:referred to as the TAT peptide), as well as the discovery and synthesisof over 100 novel CPPs 2012).

Potential therapeutics fused to CPPs have been assessed in neuronalculture systems and animal models that mimic neural injury mechanisms ina variety of disorders, including cerebral ischemia, epilepsy,Parkinson's disease and Alzheimer's diseases (Lai et al, 2005; Liu etal. 2006; Arthur et al. 2007; Colombo et al. 2007; Nagel et al. 2008;Meade et al. 2009). The use of CPPs for neurological disorders isespecially attractive due to their ability to transport cargo across theblood brain barrier and then enter into neural cells within the brainparenchyma (Aarts et al. 2002; Zhang et al. 2013). Two examples ofCPP-fused neuroprotective peptides that have entered clinical trials arethe JNK inhibitor peptide (JNKI-1D-TAT or XG-102; ARAMIS, 2012) and theNMDA receptor/postsynaptic density-95 inhibitory peptide (TAT-NR2B9c orNA-1; Dolgin, 2012), Both peptides are fused to TAT.

An important feature of any CPP is limited toxicity at clinicallyrelevant doses, and there is a great need for CPPs of limited toxicity.Similarly, there is a great need when treating neural injuries forpeptides that are neuroprotective. The structures and amino acid contentof CPPs vary wildly and it has recently been shown that the TAT peptide,the most widely used CPP used in neuroprotection experiments, appears toalso possess intrinsic neuroprotective properties. Recent studies (Xu etal, 2008; Vaslin et al. 2009; Meade et al. 2010a,b; Craig .et al. 2011)have reported that the TAT peptide displays neuroprotective actions invitro following excitotoxicity and oxygen-glucose deprivation, and invivo following cerebral ischemia in P12 rats after intraventricularinjection. While the exact mechanisms of TAT's neuroprotective actionare not fully understood, there is speculation that it interferes withNMDA receptor activation (Xu et al. 2008; Vaslin et al. 2009), althoughone study failed to detect a binding interaction (Li et al. 2008).Additionally, in an RNAi study using CPPs to deliver constructs, boththe TAT and penetratin peptides alone were shown to down-regulate MAPkinase mRNA in the lung following intratracheal administration (Moschoset al. 2007).

Neuronal or neural injuries or disorders such as migraine, stroke,traumatic brain injury, spinal cord injury, epilepsy andneurodegenerative disorders including Huntington's Disease (HD),Parkinson's Disease (PD), Alzheimer's Disease (AD) and AmyotrophicLateral Sclerosis (ALS) are major causes of morbidity and disabilityarising from long term brain or spinal cord injury. The brain injuriesgenerally involve a range of cell death processes including apoptosis,autophagy, necroptosis and necrosis, and affect neurons astrocytes,oligodentrocytes, microglia and vascular endothelial cells (collectivelyreferred to as the neurovascular unit; NVU). The damaging triggersinvolved in neural injury involve diverse pathways involving glutamateexcitotoxicity calcium overload, oxidative stress, proteolytic enzymesand mitochondrial disturbances.

As used herein, the term “stroke” includes any ischemic disorderaffecting the brain or spinal cord, e.g. thrombo-embolic occlusion in abrain or spinal cord artery, severe hypotension, perinatalhypoxia-ischaemia, a myocardial infarction, hypoxia, cerebralhaemorrhage, vasospasm, a peripheral vascular disorder, a venousthrombosis, a pulmonary embolus, a transient ischemic attack, lungischemia, unstable angina, a reversible ischemic neurological deficit,adjunct thrombolytic activity, excessive clotting conditions, cerebralreperfusion injury, sickle cell anemia, a stroke disorder or aniatrogenically induced ischemic period such as angioplasty, or cerebralischemia.

Increased extracellular levels of the neurotransmitter glutamate cancause neuronal cell death via acute and delayed damaging processedcaused by excitotoxicity. An accumulation of extracellular glutamateover-stimulates NMDA and AMPA receptors resulting in an influx ofextracellular calcium and sodium ions and the release of bound calciumfrom intracellular stores. Over-activation of NMDA receptors cart alsotrigger the production of damaging molecules (eg. nitric oxide, CLCA1;calcium-activated chloride channel regulator 1, calpain, SREBP1: sterolregulatory element binding protein-1) and signaling pathways (e.g. DAPK;death-associated protein kinase, CamKII: calcium-calmodulin-dependentprotein kinase II). In addition, glutamate-induced neuronaldepolarization and excitotoxicity can trigger further intracellularcalcium influx via voltage-gated calcium channels (VGCC, e.g. CaV2.2,CaV3.3), the sodium calcium exchanger (NCX), add-sensing ion channels(ASIC), transient receptor potential cation channels 2 and 7 (TRPM2/7)and: metabotropic glutamate receptors (mGluR).

The increase in intracellular calcium initiates a range of cell damagingevents involving phospholipases, proteases, phosphatases, kinases, NADPHoxidase and nitric oxide synthase, as well as the activation of pathwaystriggering cell death (i.e. apoptosis, autophagy, necroptosis andnecrosis).

Examples of compounds used to treat the neurodegenerative effects ofcerebral ischemia include U.S. Pat. No. 5,559,095, which describes amethod of treating ischemia-related neuronal damage usingomega-conotoxin peptides and related peptides which bind to and blockvoltage-gated calcium channels, and U.S. Pat. No. 4,684,624, whichdescribes treatment using certain opioid peptides. Further examplesinclude US 2009/0281036 that discloses the use of fusion peptides linkedto other peptides for reducing damaging effects of injuries to mammaliancells by inhibiting the interaction of NMDA receptor and NMDARinteracting proteins. Similarly, US2012/0027781 discloses the use oflinked targeting peptides and other peptides to provide neuroprotectivefunctioning. U.S. Pat. No. 6,251,854 discloses compounds that provideprotection against excitotoxic neuronal damage which are selected fromshort arginine-rich oligopeptides combined with compounds of formula 1:

Many potential neuroprotective agents also exhibit toxicity at low tomoderate doses. Many CPPs also exhibit toxicity at low to moderatedoses. There is a need thus for neuroprotective peptides which areeffective at low doses, which exhibit low cellular toxicity, and whichprovide protection against more than one type of neural injury.

This discussion of the background art is intended to facilitate anunderstanding of the present invention only. No acknowledgement oradmission that any of the material referred to is, or was, part of thecommon general knowledge as at the priority date of the application isintended.

SUMMARY OF THE INVENTION

According to one aspect of the invention, there is provided the use ofan isolated, basic (i.e cationic) amino acid-rich peptide as aneuroprotective agent. The isolated peptide may be a CPP.

As such, the invention extends to an isolated, cationic amino acid-richpeptide having neuroprotective activity. The invention extends tofunctional fragments of the peptide that exhibit neuroprotectiveactivity.

The peptide may have a net charge of 8 or higher at pH 7, preferably 10or higher at pH 7, most preferably 11 or higher at pH 7.

The peptide may be a non-naturally occurring peptide. As such, thepeptide of the invention may be a man-made peptide.

The cationic (i.e. basic) amino acid residues forming part of thepeptide may be arginine or lysine. Thus, the peptide may bearginine-rich. Alternatively, or additionally, the peptide may belysine-rich, and/or tryptophan-rich.

The peptide may be between 10 amino acids and 100 amino acids in length,preferably between 10 amino acids and 32 amino acids.

The peptide may have the sequence:

X_((K))—Z_((N))—X_((K))—Z_((N))—X_((K))

wherein X may be any naturally occurring or synthetic amino acid,including a cationic (i.e. basic) amino acid residue;

K is an integer between 1 and 5;

Z is a basic (i.e. cationic) amino acid residue; and

N is an integer between 1 and 30.

Substitution at “X” positions with amino acids which do not decrease theneuroprotective effects of the neuroprotective peptides are preferred.In one embodiment, Z may be arginine. In another embodiment, Z may belysine. The peptide may include at least one contiguous arginine-richsegment. In other embodiments, the peptide may include a non-contiguousarginine-rich segment.

The basic (i.e. cationic) amino acid residues may be included at a ratioof at least 30% of the peptide or segment, preferably at least 40%,preferably at least 50%, more preferably at least 60%, in some cases ashigh as 100% of the peptide or segment. The peptide may have an argininecontent of more than 20% of the amino acid content of the peptide,preferably more than 30%, more than 40%, more than 50%, more than 60%,more than 70% more than 80%, more than 90, more than 95%, more than 99%,or most preferably 100%. In certain embodiments of the invention, thepeptide may have a combined arginine and lysine content of more than40%, more than 50%, more than 60%, more than 70% more than 80%, morethan 90, more than 95%, more than 99%, or most preferably 100%.

The peptide may include a plurality of single cationic amino acidresidues, such as arginine residues, interspersed by other amino acidresidues, in particular, it may be interspersed with basic (i.e.cationic) amino acid residues, such as lysine, and/or tryptophan. Thepeptide may include repeats of arginine residues in adjacent positions,such as RR, or RRR, or RRRR, or higher order repeats, and may beinterspersed between other amino acids, or between stretches of aminoacids.

As such, the peptide may be comprised completely of cationic aminoacids. In one embodiment, the peptide is comprised completely ofarginine residues.

According to an aspect of the invention, there is provided use of anisolated peptide of 10 to 32 amino acid residues in length for thetreatment of neural injury, wherein the isolated peptide comprises atleast 10 to 22 residues.

In a preferred embodiment of the invention, the peptide may be anisolated peptide of 10 to 32 amino acid residues in length for thetreatment of neural injury, wherein the isolated peptide comprises atleast 10 to 22 arginine residues.

The isolated peptide may have an arginine residue content of at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, or at least 99%.

The isolated peptide may be a poly-arginine peptide comprising 10 to 18arginine residues. In a preferred embodiment, the isolated peptide isR12, R15, or R18, most preferably R15.

Specifically, the isolated peptide may comprise any one or more of thepeptides selected from the group consisting of SEQ. ID. NOS. SEQ, ID.NOS. 7, 8, 9, 10, 11, 12, 13, 16, 30, 31, 32, 33, 34, 35, 36, 37, andfunctional fragments and analogues thereof having neuroprotectiveactivity.

The isolated peptide may include at least one poly-arginine segmentcomprising at least 4 contiguous arginine residues, preferably at leasttwo poly-arginine segments, more preferably three poly-argininesegments.

The isolated peptide may be penetratin (SEQ. ID. NO. 22).

The isolated peptide may comprise a mix of protamine derivatives. Themix of protamine derivatives may comprise protamine sulphate.

The isolated peptide may affect the endocytotic processes of the cell;affect the function of cell surface receptors to result in reducedcellular calcium influx; interact with and/or stabilises the outermitochondrial membrane to preserve mitochondrial function; or inhibit,downregulate, or affect the calcium-dependent pro-protein convertaseenzyme furin.

The isolated peptide may be a synthetic peptide or a man-made peptide.The isolated peptide may be included within, or fused to, otherpolypeptides. The isolated peptides may be fused to one another with atleast one linker sequence

The isolated peptide may exhibit neuroprotective activity at IC50 levelsof less than 50 μM, preferably less than 20 μM, most preferably lessthan 10 μM.

The invention extends to the use of an isolated peptide comprising anyone or more of SEQ. ID. NOS. SEQ. ID. NOS. 7, 8, 9, 10, 11, 12, 13, 16,30, 31, 32, 33, 34, 35, 36, 37, and functional fragments or analoguesthereof having neuroprotective activity, in the manufacture of apharmaceutical composition or medicament for treating or preventingneural injury.

The pharmaceutical composition or medicament may be used for thetreatment or prevention of a neural injury, the pharmaceuticalcomposition or medicament including the isolated peptide of theinvention or the polynucleotide sequence of the invention.

The pharmaceutical composition or medicament may be used in thetreatment or prevention of ischemia, perinatal hypoxi-ischemia,Alzheimer's disease, Huntington's Disease, Multiple Sclerosis,Parkinson's disease, amyotrophic lateral sclerosis, stroke, peripheralneuropathy, spinal cord injury, or epilepsy.

The pharmaceutical composition or medicament may comprise apharmaceutically acceptable carrier, adjuvant, or vehicle.

According to another aspect of the invention there is provided a methodof treating a neural injury or promoting survival of neurons, the methodincluding the steps of administering to a patient in need thereof apharmaceutically acceptable and/or pharmaceutically effective amount ofthe peptide of the invention or the pharmaceutical composition ormedicament of the invention.

According to a still further aspect of the invention there is provided amethod for inhibiting neuronal cell death in a subject comprisingadministering to a subject in need of such treatment a pharmaceuticallyacceptable and/or pharmaceutically effective amount of the peptide ofthe invention or the pharmaceutical composition or medicament of theinvention.

Administration may be from 0.001 mg/kg to 50 mg/kg.

The invention extends to kit comprising the pharmaceutical compositionor medicament of the invention in one or more container(s) and aninstruction manual or information brochure regarding instructions and/orinformation with respect to application of the pharmaceuticalcomposition.

More specifically, the peptide may be any one or more of the peptideshaving the sequences set forth as SEQ, ID, NOS. SEQ. ID. NOS. 7, 8, 9,10, 11, 12, 13, 16, 30, 31, 32, 33, 34, 35, 36, 37, or may includesequences having at least 60%, preferably 70%, more preferably 80%, evenmore preferably 90%, yet more preferably 99% or higher sequence identityto said peptides, such peptides having neuroprotective activity.

The peptide may comprise any length of poly-arginine peptide between R8and R22, or repeats thereof. In one embodiment, the peptide is selectedfrom the group comprising R8, R9, R10, R11, R12, R13, R14, R15, R16,R17, and R18. In one preferred embodiment, the peptide is R10. Inanother preferred embodiment, the peptide is R12. In yet anotherpreferred embodiment, the peptide is R15. In another preferredembodiment, the peptide is R18.

The use of the peptide may include the use of a mixture of any two ormore of the peptides of the invention, particularly SEQ. ID. NOs 32 to36, in the treatment or prevention of a neural injury.

In another embodiment, the use may include using the peptide of SEQ. ID.NO. 37 in the treatment or prevention of a neural injury, or the use ofSEQ. ID. NO. 37 with any of the peptides of SEQ. ID. NOS. 32 to 36.

In another aspect of the invention, there is provided the isolatedpeptides of SEQ ID NOs 32 to 37, including sequences having at least60%, 70%, 80%, 90%, even 99% or higher sequence identity to saidpeptides, such peptides having neuroprotective activity.

The peptides may be a commercially available mixture of protaminepeptides isolated from salmon sperm.

As such, the peptides may be in the form of a mixture comprisingprolamine sulphate, as described in the European. Medicines Agency“Assessment Report for Protamine containing medicinal products”,Procedure no EMEA/H/A-5(3)/1341 published 15 Nov. 2012, the contents ofwhich are incorporated herein by way of reference only.

Table 1 shows a summary of different forms of salmon sperm protamine.The protamine peptide sequences (salmon) are present in protaminesulphate for clinical use or from the SwissProt database. As used inthis specification, the term “Protamine” refers to a commerciallyavailable injectable form or protamine sulphate, which comprises amixture of the peptides of SEQ. ID. NOs 32 to 35.

The peptides may be present in the following percentage proportions inthe mixture of peptides:

SEQ. ID. NO Identifier Percentage 32 Ptm1 15-25 33 Ptm2 27-37 34 Ptm320-30 35 Ptm4 15-25

In a preferred embodiment, the peptides are present in the followingpercentage proportions in the mixture of peptides:

SEQ. ID. NO Identifier Percentage 32 Ptm1 18-22 33 Ptm2 30-35 34 Ptm321.5-28  35 Ptm4 19-23

In a most preferred embodiment, the peptides are present in thefollowing percentage proportions in the mixture of peptides:

SEQ. ID. NO Identifier Percentage 32 Ptm1 20.1 33 Ptm2 33.5 34 Ptm3 25.135 Ptm4 21.3

More particularly, the mixture of peptides may be a mixture of thepeptides of SEQ. ID. NOs 32, 33, 34, and 35, commercially available asprotamine sulphate (Salmon), manufactured e.g. by Sanofi Aventis.

As such, the mixture of peptides may be admixed with sodium chloride,hydrochloric acid, sodium hydroxide and water.

The mixture of peptides may be in a delivery formulation that isinjectable or administrable intravenously.

The peptides may be present in the delivery formulation in aconcentration within a range of 0.1 mg/ml to 100 mg/ml, preferably 1mg/ml to 20 mg/ml, most preferably 10 mg/ml.

In use, the delivery formulation, when in the form of an injectable orintravenous formulation, may be administered by slow intravenousinjection over a period of between 1 and 30 minutes, preferably between5 and 20 minutes, most preferably over a period of 10 minutes.

The peptide may have cell-penetrating activity. As such, the peptide maybe a CPP.

The peptide may include repeats of arginine residues in adjacentpositions, such as. RR, or RRR, or RRRR, or higher order repeats, andmay be interspersed between other amino acids, or between stretches ofamino acids.

According to a further aspect of the invention, there is provided theuse of the peptide of the invention in the manufacture of apharmaceutical composition or medicament for the treatment of a neuralinjury. The invention extends to the use of mixtures of peptides of theinvention in the manufacture of a medicament for the treatment of neuralinjury.

According to a still further aspect of the invention, there is providedthe use of the peptide or pharmaceutical composition of the invention toaffect the function of cell surface receptors associated with calciuminflux, more specifically to interact with the NMDA, AMPA, VGCCs, NCX,TRMP2/7, ASIC and mGlu receptors, more specifically still to result inreduced cellular calcium influx. In another embodiment of the invention,there is provided the use of the peptide or pharmaceutical compositionof the invention to interact with and/or stabilise the outermitochondrial membrane and thereby help to preserve mitochondrialfunction. According to a still further aspect of the invention, there isprovided the use of the peptide or pharmaceutical composition of theinvention to inhibit, downregulate, or affect the calcium-dependentpro-protein convertase enzyme furin.

According to another aspect of the invention, there is provided apharmaceutical composition or medicament for the treatment of a neuralinjury, the pharmaceutical composition or medicament including theisolated peptide of the invention, or any one or more of the isolatedpeptides of the invention.

The pharmaceutical composition or medicament may comprise apharmaceutically acceptable carrier, adjuvant, or vehicle

According to a still further aspect of the invention, there is provideda method of treating a neural injury, the method including the steps ofadministering to a patient in need thereof a pharmaceutically acceptableand/or pharmaceutically effective amount of the peptide of theinvention. The patient may be administered a physiologically acceptableamount of the peptide of the invention.

According to a still further aspect of the invention there is provided amethod for inhibiting neuronal cell death in a subject comprisingadministering to a subject in need of such treatment a neuroprotectivepeptide in an amount effective to inhibit neuronal cell death in thesubject, where the neuroprotective peptide is any one or more of thepeptides of the invention.

The patient may be administered a physiologically acceptable amount of amixture of any two or more of the peptides of the invention.

More particularly, the patient may be administered a mixture of any twoor more peptides selected from the group comprising SEQ. ID. NOs 32, 33,34, 35, 36, or 37. In one particular aspect of the invention, thepatient may be administered a mixture comprising SEQ. ID. NOs 32, 33, 34and 35. In other combinations, the patient may be administered anycombination of peptides selected from the group consisting of SEQ. ID.NOs 32, 33, 34 and 35, together with the peptide of SEQ. ID. NO. 36. Ina particular embodiment, the patient may be administered SEQ. ID. NO.35.

The peptide of the invention may provide neuroprotective activityagainst antagonists of neurotransmitter receptors. The neurotransmitterreceptors may be receptors that are bound by, interact with, or areaffected by NMDA, glutamate, kainic acid, or ischemic processes. Thepeptide of the invention may be included within other polypeptides ormay be fused to other polypeptides. The peptide of the invention may belinked or fused at either the N- or C-termini of such otherpolypeptides. The peptide of the invention may be linked or fused tosuch other polypeptides so as to display the peptides of the inventionin a conformation suitable for treating a neural injury.

Alternatively, or additionally, one or more of the peptides of theinvention may be fused to one another with a linker sequence. The linkersequence may comprise any sequence of amino acids, including, but notlimited to basic/cationic amino acid-rich linker sequences.Alternatively, or additionally, the linker sequences may be cleavablelinkers. In one embodiment, the linker may be one or more MMP-typelinkers, calpain, caspase or tPA linkers. MMP-type linkers are definedas the peptide sequence recognized and cleaved by matrixmetalloproteinases (MMPs), Similarly a calpain, caspase or tPA linker isa peptide sequence recognized and cleaved by these protease enzymes(i.e. calpain, caspase or tPA, respectively). The peptides of theinvention may also be fused to other peptides that are to be transportedto the site of a neural injury or are to be transported intracellularlyinto or within neural cells.

The peptides of the invention may also be linked to ancillary peptidesthat can bind to or interact with enzymes detrimental to neuralfunction, so that the ancillary peptides can function as competitiveinhibitors of the enzymes following transportation across the cellmembrane. The ancillary peptides may be selected from tPA, calpain, andMMP, and may be linked to the peptide of the invention using a cleavablelinker such as a caspase sequence, so that the tPA, calpain or MMP maybe liberated from the peptide of the invention and may then function asa competitive inhibitor intracellularly for enzymes detrimental toneural function.

As such, the invention extends to a peptide of the invention linked to acaspase cleavage site, itself in turn linked to calpain, tPA or MMP.

The peptides of the invention may exhibit neuroprotective activity atIC50 levels of less than 10 μM, preferably less than 5 μM, preferablyless than 1 μM, in some cases as low as, or lower than, 0.2 μM, even aslow as 0.1 μM.

As mentioned before, the peptide may include repeats of the peptidesequences of the invention, or functional fragments thereof, i.e.fragments that exhibit neuroprotective activity.

Accordingly, one aspect of the invention provides an isolatedpolypeptide having included therein a peptide segment exhibitingneuroprotective effects, the peptide segment being between 8 and 100amino acid residues in length, wherein the neuroprotective peptide isselected from peptide segments having a basic/cationic amino add contentof more than 20% of the length of the peptide segment, preferably morethan 30%, more than 40%, more than 50%, more than 60%, more than 70%more than 80%, more than 90, more than 95%, more than 99%, or mostpreferably 100%.

According to another aspect of the invention, there is provided anisolated polynucleotide sequence that encodes a peptide of theinvention, sequences complementary to the isolated polynucleotidesequence, and sequences having at least 60%, 70%, 80%, 90%, 95%, 99%, or100% homology with the isolated polynucleotide sequences. Thepolynucleotide sequence may be one or more isolated sequences and may besequences that hybridize under stringent conditions with thepolynucleotide sequences of the invention. The polynucleotide sequencesmay be non-naturally occurring polynucleotide sequences or DNA. As such,they may include man-made, artificial constructs, such as cDNA. Alsoincluded in the invention are vectors, such as expression vectors, whichinclude the isolated foregoing isolated nucleic acids, as well as cellstransformed with such vectors or DNA sequences. The polynucleotidesequence may encode. protamine. The polynucleotide sequences may be thesequence of SEQ. ID. NO. 38.

In another aspect of the invention, there is provided the isolatedpeptides selected from the group consisting of SEQ. ID. NOS. 7, 8, 9,10, 11, 12, 13, 16, 30, 31, 32, 33, 34, 35, 36, and 37, includingsequences having at least 60%, preferably 70%, more preferably 80%, evenmore preferably 90%, yet more preferably 99% or higher sequence identityto said peptides, such peptides having neuroprotective activity.

The use of the isolated nucleic acids, vectors, or cells in thepreparation of a pharmaceutical formulation or medicament also isprovided.

The present invention furthermore provides kits comprising theabovementioned pharmaceutical composition (in one or more container(s))in at least one of the above formulations and an instruction manual orinformation brochure regarding instructions and/or information withrespect to application of the pharmaceutical composition.

In one embodiment, a pharmaceutical composition comprising the peptideof the invention as defined above is for use in the treatment ofischemia, Alzheimers disease, Parkinson's disease, amyotrophic lateralsclerosis, stroke, peripheral neuropathy, spinal cord injury orepilepsy.

In another embodiment, the present invention provides a method forpromoting survival of neurons comprising the step of contacting neuronswith the peptides of the invention, or combinations thereof. Preferably,the method is performed in vitro. The invention, in another aspectthereof, provides a method and composition for protecting blood brainbarrier endothelial cells from OGD or ischemia.

These findings demonstrate that the peptides of the invention have theability to, and can be used in methods to, inhibit or ameliorateneurodamaging events/pathways associated with excitotoxic and ischemicinjuries. Also, as shown by the effects of protamine and protaminederivatives in pre-insult exposure trials contained herein, a new keyfinding was that protamine treatment of neurons 1 to 4 hours beforeglutamate or OGD exposure can induce a neuroprotective response byreducing cell death. This is significant because there are a number ofcerebrovascular (e.g. carotid endarterectomy) and cardiovascular (e.g.coronary artery bypass graft) surgical procedures where there is a riskpatients can suffer cerebral ischemia or a stroke resulting in braininjury.

Therefore, the method of the invention extends to the administering ofthe at least one peptide, medicament, or pharmaceutical composition ofthe invention in a window of 0.25 hours to 4 hours, preferably 0.5 to 3hours, most preferably 1 to 2 hours before such a procedure to protectthe brain against any such cerebral ischemic event,

The invention extends thus to the use of the at least one peptide of theinvention in treating or preventing neural injury, cerebrovascularinsults or injury, cardiovascular insults or injury, or surgicalprocedures where patients may be at risk of suffering cerebral ischemiaor a stroke,

Minor modifications of the primary amino acid sequence of the sequencesof the invention disclosed herein may result in proteins that havesubstantially equivalent or enhanced activities. These modifications maybe deliberate, as through site-directed mutagenesis, or may beaccidental, such as through mutation of hosts that are protamine- orLMWP-producing organisms. All of these modifications are included withinthe scope of the invention as long as the neuroprotective activity isretained.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features of the present invention are more fully described inthe following description of several non-limiting embodiments includedsolely for the purposes of exemplifying the present invention. Thefollowing description is not a restriction on the broad summary,disclosure or description of the invention as set out above and is madewith reference to the accompanying drawings in which the term“Protamine” or “Ptm” refers to commercially available protamine sulphate(manufactured commercially by several manufacturers, including SanofiAventis, typically as specified in Hoffman, 1990), while “Ptm1” to “Ptm”5 refer to SEQ. ID. NOs 32, 33, 34, 35, and 36, respectively, and inwhich:

FIG. 1a shows the results of the glutamic acid excitotoxicity model;concentration of peptide in μM. A: Neuronal viability 24 hours followingglutamic acid exposure and treatment with CPPs, positive controlpeptides (JNKI-1D-TAT/PYC36L-TAT) and glutamate receptor blockers(Blkrs; 5 μM: MK801/5 μM: CNQX). MTS data were expressed as percentageneuronal viability with no insult control taken as 100% viability(mean±SEM; n=4-6.; *P<0.05).

FIG. 1b shows further results of the Glutamic acid excitotoxicity model;concentration of peptide in μM. Neuronal viability 24 hours followingglutamic acid exposure and treatment with TAT-L and TAT-D peptides. MTSdata were expressed as percentage neuronal viability with no insultcontrol taken as 100% viability (mean±SEM; n=4-6; *P<0.05).

FIG. 1c shows further results from the glutamic acid excitotoxicitymodel. Specifically, the efficacy of peptides when washed out prior toglutamic acid insult; concentration of peptide in μM. Neuronal viability24 hours following glutamic acid exposure when CPPs were washed-outprior to insult. MTS data were expressed as percentage neuronalviability with no insult control taken as 100% viability (mean±SEM;n=4-6; *P<0.05).

FIG. 1d shows further results from the glutamic acid excitotoxicitymodel. Efficacy of peptides when added after the glutamic acid insult.Neuronal viability 24 hours following glutamic acid exposure when Arg-9and Arg-12 peptides and control peptides JNKI-1D-TAT and NR29c wereadded 0 or 15 minutes post-insult; in this experiment glutamic acidexposure resulted in less cell death in controls than in otherexperiments (60% vs 95%). MTS data were expressed as percentage neuronalviability with no insult control taken as 100% viability (mean±SEM;n=4-6, *P<0.05).

FIG. 1e shows further results from the glutamic acid excitotoxicitymodel; concentration of peptide in μM. A: Neuronal viability 24 hoursfollowing glutamic acid exposure and treatment with R9, R12, R15, R18and R9/tPA/R9. MTS data were expressed as percentage neuronal viabilitywith no insult control taken as 100% viability (mean±SEM; n=4; *P<0.05).

FIG. 1f shows further results from the glutamic acid excitotoxicitymodel; concentration of peptide in μM. Neuronal viability 24 hoursfollowing glutamic acid exposure and treatment with R9, E9/R9, DAHK andPTD4. MTS data were expressed as percentage neuronal viability with noinsult control taken as 100% viability (mean±SEM; n=4; *P<0.05).

FIG. 1g shows further results from the glutamic acid excitotoxicitymodel; concentration of peptide μM. Neuronal viability 24 hoursfollowing glutamic acid exposure and treatment with R9 and NR29c andcontrol peptide PCY36 (PYC36L-TAT). MTS data were expressed aspercentage neuronal viability with no insult control taken as 100%viability (mean±SEM; n =4; *P<0.05).

FIG. 1h shows further results from the glutamic acid excitotoxicitymodel; milder insult; concentration of peptide μM. Neuronal viability 24hours following glutamic acid exposure and treatment with R9, R12 andNR29c and control peptide JNK (JNKI-1-TAT). MTS data were expressed aspercentage neuronal viability with no insult control taken as 100%viability (mean±SEM; n=4; *P<0.05).

FIG. 1i shows further results from the glutamic acid excitotoxicitymodel: milder insult; concentration of peptide μM. Neuronal viability 24hours following glutamic acid exposure and treatment with R1, R3, R6,R9, R12 and NR29c control peptide. MTS data were expressed as percentageneuronal viability with no insult control taken as 100% viability(mean±SEM; n=4; *P<0.05).

FIG. 1j shows further results from the glutamic acid excitotoxicitymodel; concentration of peptide=5 μM. Neuronal viability 24 hoursfollowing glutamic acid exposure and treatment with R1, R3, R9 old(Mimotopes), R9 new (China peptides), R12. MTS data were expressed aspercentage neuronal viability with no insult control taken as 100%viability (mean±SEM; n=4; *P<0.05).

FIG. 2 shows the results of a kainic acid excitotoxicity model;concentration of peptide in μM. Neuronal viability 24 hours followingkainic acid exposure and treatment with CPPs, positive control peptides(JNKI-1D-TAT/PYC36L-TAT) and glutamate receptor blockers (Blkrs; 5 μM:MK801/5 μM: CNQX). MTS data were expressed as percentage neuronalviability with no insult control taken as 100% viability (mean±SEM; n=4;*P<0.05).

FIG. 3a shows the results of an in vitro ischemia model. Peptidespresent during in vitro ischemia and at 50% dose after ischemia:concentration of peptide in μM. Neuronal viability 24 hours following invitro ischemia and treatment with CPPs, positive control peptide(PYC36L-TAT) and glutamate receptor blockers (Blkrs; 5 μM: MK801/5 μM:CNQX). MTS data were expressed as percentage neuronal viability with noinsult control taken as 100% viability (mean±SEM; n=4; *P<0.05).

FIG. 3b shows further results of an in vitro ischemia model. Peptidespresent after in vitro ischemia: concentration of peptide in μM.Neuronal viability 24 hours following in vitro ischemia and treatmentwith R9, R12, R15 and R18. MTS data were expressed as percentageneuronal viability with no insult control taken as 100% viability (meanSEM; n=4; *P<0.05).

FIG. 3c shows further results of an in vitro ischemia model. R9 peptidepresent during in vitro ischemia and at 50% dose after ischemia: Peptidedose for bEND3 cells was 10 μM during/5 μM post-in vitro ischemia.Peptide dose for SH-5YSY cells was 5 μM during/2.5μM post-in vitroischemia. Cell viability 24 hours following in vitro ischemia (MTS datamean±SEM; n =4; *P<0.05).

FIG. 4 shows neuronal viability following exposure of cultures todifferent peptide concentrations. Peptide concentration shown in μM.Neuronal viability 24 hours following exposure with R3, R6, R9, R12,R15, R18, JNK (JNKI-1D-TAT) and NR29c peptides. Cell viability dataexpressed as MTS absorbance at 490 nm (mean±SEM; n=4).

FIG. 5 shows the results of the initial animal model pilot trial, whichshows the efficacy of R9D peptide in rat permanent middle cerebralartery occlusion (MCAO) stroke model. R9D peptide was administeredintravenously 30 min post-MCAO. Infarct volume (brain injury) wasmeasured 24h post-MCAO (mean±SD).

FIG. 6 shows a dose response study using R9, R10, R11, R12, R13 and R14in the glutamate model. Mean±SEM: N =4; * P<0.05. (Peptide concentrationin μM).

FIG. 7 shows a dose response study using R9D, R13, R14 and R15 in theglutamate model Mean±SEM: N =4; * P<0.05. (Peptide concentration in μM).

FIG. 8 shows a dose response study using. R6, R7, R8, and R9 in theglutamate model. Mean±SEM: N =4; * P<0.05. (Peptide concentration inμM).

FIG. 9 shows the results of the glutamic acid excitotoxicity model;concentration of protamine in μM. Treatment of neuronal cultures withprotamine was for 15 minutes prior to glutamic acid exposure (100 μM),which was for 5 minutes at 37° C. Neuronal viability 24 hours followingglutamic acid exposure and treatment with different protamineconcentrations or no treatment (Glut control). MTS data were expressedas percentage neuronal viability with no insult control taken as 100%viability (mean±SD; n=4-6; *P<0.05).

FIG. 10 shows the results of the glutamic acid excitotoxicity model;concentration of protamine in μM. Treatment of neuronal cultures withprotamine was for 15 minutes prior to and during glutamic acid exposure(100 μM), which was for 5 minutes at 37° C. Neuronal viability 24 hoursfollowing glutamic acid exposure and treatment with different protamineconcentrations or no treatment (Glut control). MTS data were expressedas percentage neuronal viability with no insult control taken as 100%viability (mean±SD; n=4-6; *P<0.05).

FIG. 11 shows the results of the glutamic acid excitotoxicity model;concentration of protamine in μM. Treatment of neuronal cultures withprotamine was for 5 or 10 minutes prior to glutamic acid exposure (100μM), (5 minutes at 37° C.) only. Neuronal viability 24 hours followingglutamic acid exposure and treatment with different protamineconcentrations or no treatment (Glut). MTS data were expressed aspercentage neuronal viability with no insult control taken as 100%viability (mean±SD; n=4-6; *P<0.05).

FIG. 12 shows the results of the glutamic acid excitotoxicity model;concentration of protamine and low molecular weight protamine (LMWP) inμM. Treatment of neuronal cultures with protamine or LMWP was for 15minutes prior to and during glutamic acid exposure (100 μM), which wasfor 5minutes at 37° C. Neuronal viability 24 hours following glutamicacid exposure and treatment with different protamine or LMWPconcentrations or no treatment (Glut control). MTS data were expressedas percentage neuronal viability with no insult control taken as 100%viability (mean±SD; n=4-6; *P<0.05).

FIG. 13 shows the results of the glutamic acid excitotoxicity model;concentration of protamine and low molecular weight protamine (LMWP) inμM. Treatment of neuronal cultures with protamine or LMWP was for 15minutes prior to and during glutamic acid exposure (100 μM), which wasfor 5 minutes at 37° C. Neuronal viability 24 hours following glutamicacid exposure and treatment with different protamine or LMWPconcentrations or no treatment (Glut control). MTS data were expressedas percentage neuronal viability with no insult control taken as 100%viability (mean±SD; n=4-6; *P<0.05).

FIG. 14 shows the results of the glutamic acid excitotoxicity model;concentration of protamine and LMWP in μM. Treatment of neuronalcultures with protamine was for 10 minutes immediately before, or 1 or 2hours before glutamic acid exposure (100 μM; 5 minutes at 37°C.) only.Neuronal viability 24 hours following glutamic acid exposure andtreatment with different protamine or LMWP concentrations or notreatment (Glut Control). MTS data were expressed as percentage neuronalviability with no insult control taken as 100% viability (mean±SD;n=4-6; *P<0.05).

FIG. 15 shows the results of the glutamic acid excitotoxicity model;concentration of protamine and protamine peptides 1 to 5 (“Ptm 1 to 5”)in μM. Treatment of neuronal cultures with protamine and protaminepeptides was for 15 minutes prior to glutamic acid exposure (100 μM),which was for 5 minutes at 37° C. Neuronal viability 24 hours followingglutamic acid exposure and treatment with different protamine andprotamine peptides concentrations or no treatment (Glut control). MTSdata were expressed as percentage neuronal viability with no insultcontrol taken as 100% viability (mean±SD; n=4-6; *P<0.05).

FIG. 16 shows the results of the oxygen-glucose deprivation (OGD) model;concentration of protamine in μM. Treatment of neuronal cultures withprotamine was immediately after 50 minutes of OGD and until experimentend (24 h). Neuronal viability 24 hours following OGD and treatment withdifferent protamine concentrations or no treatment (OGD). MTS data wereexpressed as percentage neuronal viability with no OGD control taken as100% viability (mean±SD; n=4-6; *P<0.05).

FIG. 17 shows the results of the oxygen-glucose deprivation (OGD) model;concentration of protamine in μM. Treatment of neuronal cultures withprotamine was immediately after 50 minutes of OGD and until experimentend (24 h). Neuronal viability 24 hours following OGD and treatment withdifferent protamine concentrations or no treatment (OGD). MTS data wereexpressed as percentage neuronal viability with no OGD control taken as100% viability (mean SD; n=4-6; *P<0.05).

FIG. 18 shows the results of the oxygen-glucose deprivation (OGD) model;concentration of protamine and LMWP in μM. Treatment of neuronalcultures with protamine or LMWP was for 10 minutes, 1 hour before 50minutes of OGD. Neuronal viability 24 hours following OGD andpre-treatment with different protamine concentrations or no treatment(OGD). MTS data were expressed as percentage neuronal viability with noOGD control taken as 100% viability (mean±SD; n=4-6; *P<0.05).

FIG. 19 shows the results of the oxygen-glucose deprivation (OGD) model;concentration of protamine in μM. Treatment of neuronal cultures withprotamine was for 15 or 30 minutes, after 50 minutes of OGD. Neuronalviability 24 hours following OGD and post-treatment with differentprotamine concentrations or no treatment (OGD). MTS data were expressedas percentage neuronal viability with no OGD control taken as 100%viability (mean±SD; n=4-6; *P<0.05).

FIG. 20 shows the results of the oxygen-glucose deprivation (OGD) model;concentration of protamine and LMWP in μM. Treatment of neuronalcultures with protamine or LMWP was for 15 minutes, after 50 minutes ofOGD. Neuronal viability 24 hours following OGD and post-treatment withdifferent protamine or LMWP concentrations or no treatment (OGD). MTSdata were expressed as percentage neuronal viability with no OGD controltaken as 100% viability (mean±SD; n=4-6; *P<0.05).

FIG. 21 shows the results of the oxygen-glucose deprivation (OGD) model;concentration of protamine and protamine peptides in μM (5 μM).Treatment of neuronal cultures with protamine was for 15 minutes, after45 minutes of OGD. Neuronal viability 24 hours following OGD andpost-treatment with different protamine and protamine peptidesconcentrations or no treatment (OGD). MTS data were expressed aspercentage neuronal viability with no OGD control taken as 100%viability (mean±SD; n=5; *P<0.05).

FIG. 22 shows the results of the oxygen-glucose deprivation (OGD) model;concentration of protamine, protamine and low molecular weight protamine(LMWP) peptides in μM (5 μM). Treatment of neuronal cultures withprotamine was for 15 minutes, after 45 minutes of OGD. Neuronalviability 24 hours following OGD and post-treatment with differentprotamine and protamine peptides concentrations or no treatment (OGD).MTS data were expressed as percentage neuronal viability with no OGDcontrol taken as 100% viability (mean±SD; n=4-6; *P<0.05).

FIG. 23 shows the results of the oxygen-glucose deprivation (OGD) modelusing brain endothelial cells (bEND 3 cells); concentration of protaminein μM. Treatment of bEND3 cultures with protamine was from 15 minutesimmediately before 3 different OGD durations (2 h15 min, 2 h30 min and 2h45 min) and until experiment end (24h). Cell viability was measured 24hours following OGD. MTS data were expressed as percentage neuronalviability with no OGD control taken as 100% viability (mean±SD; n=4-6;*P<0.05).

FIG. 24 shows the results of exposure of protamine peptides to brainendothelial cells (bEND3 cells); concentration of protamine 1 μM.Protamine peptides 1-5 (Ptm1-Ptm5), protamine sulphate (Ptm), and lowmolecular weight protamine (LMWP) added to bEND3 cell cultures for 15min immediately before OGD (2h30min duration). Cell viability assessed24 hours after OGD using MTS assay. MTS data were expressed asabsorbance values at 490mm (mean±SD; n=4-6; *P<0.05).

FIG. 25 shows the results of exposure of protamine to brain endothelialcells (bEND3 cells); concentration of protamine in μM. Treatment ofbEND3 cultures with protamine was for 0.5, 1 or 2 hours. Cell viability2 hours following protamine exposure or no treatment (Control). MTS datawere expressed as absorbance values at 490mm (mean±SD; n=4-6; *P<0.05).

FIG. 26 shows the results of exposure of protamine to brain endothelialcells (bEND3 cells); concentration of protamine in μM. Treatment ofbEND3 cultures with protamine was for 2 hours. Cell viability 2 hoursfollowing protamine exposure or no treatment (Control). MTS data wereexpressed as absorbance values at 490 mm (mean±SD; n=4-6; *P<0.05).

FIG. 27 shows neuroprotective effects of the R12, R15, R18 and protamine(Ptm) peptides in permanent middle cerebral artery occlusion (MCAO)stroke model when administered intravenously 30 min after occlusion.Peptide dose was 1 μmol/kg (600 μl: IV) and infarct assessment was at 24h after MCAO (mean±SE; n=8-10; *P<0.05). Animal treatments wererandomized and all procedures were performed blinded to treatment.

FIG. 28 shows neuroprotective efficacy of R9, R12, R15 and R18 in theglutamate excitotoxicity model when peptides present in neuronalcultures only during 5-min glutamic acid exposure. Neuronal viabilitymeasured 20-24 h following glutamic acid. Concentration of peptide inμM. MTS data were expressed as percentage neuronal viability with noinsult control taken as 100% viability (mean±SD; n=4;*P<0.05).

FIG. 29 shows neuroprotective efficacy of R9, R12, R15 and R18 in theglutamate model when peptides present in neuronal cultures for 10 minonly prior to glutamic acid exposure. Neuronal viability measured 20-24h following glutamic acid. Concentration of peptide in μM. MTS data wereexpressed as percentage neuronal viability with no insult control takenas 100% viability (mean±SD; n=4; *P<0.05).

FIG. 30 shows neuroprotective efficacy of R12 and R15 in the glutamatemodel when peptides present in neuronal cultures for 10 min only at 1 to5 h before glutamic acid exposure. Neuronal viability measured 20-24 hfollowing glutamic acid. Concentration of peptide in μM. MTS data wereexpressed as percentage neuronal viability with no insult control takenas 100% viability (mean±SD; n=4; *P<0.05).

FIG. 31 shows neuroprotective efficacy of R9, R12, R15 and R18 in theoxygen-glucose deprivation (OGD) model when peptides added to neuronalcultures immediately after OGD and removed after 15 min. Neuronalviability measured 20-24 hours following OGD. Concentration of peptidein μM. MTS data were expressed as percentage neuronal viability with noinsult control taken as 100% viability (mean±SD; n=4-6; *P<0.05).

FIG. 32 shows neuroprotective efficacy of R9, R12, R15 and R18 in theoxygen-glucose deprivation when peptides present in neuronal culturesonly for 10 min at 1 to 3 h before OGD. Neuronal viability measured20-24 hours following OGD. Concentration of peptide in μM. MTS data wereexpressed as percentage neuronal viability with no insult control takenas 100% viability (mean±SD; n=4-6; *P<0.05).

FIG. 33 shows neuroprotective efficacy of PTD4, E9/R9 and R9 in theglutamate excitotoxicity model when peptides present in neuronalcultures for 15 min before and during 5-min glutamic acid exposure.Neuronal viability measured 20-24 hours following glutamic acid.Concentration of peptide in μM. MTS data were expressed as percentageneuronal viability with no insult control taken as 100% viability(mean±SD; n=4; *P<0.05).

FIG. 34 shows neuroprotective efficacy of XIP and PYC36-TAT in theglutamate excitotoxicity model when peptides present in neuronalcultures for 15 min before and during 5-min glutamic acid exposure.Neuronal viability measured 20-24 hours following glutamic acid.Concentration of peptide in μM. MTS data were expressed as percentageneuronal viability with no insult control taken as 100% viability(mean±SD; n=4; *P<0.05).

FIG. 35 shows neuroprotective efficacy of NCXBP3 in the glutamateexcitotoxicity model when peptide present in neuronal cultures for 15min before and during 5-min glutamic acid exposure. Neuronal viabilitymeasured 20-24 hours following glutamic acid. Concentration of peptidein μM. MTS data were expressed as percentage neuronal viability with noinsult control taken as 100% viability (mean±SD; n=4; *P<0.05).

FIG. 36 shows neuroprotective efficacy of K10, R10 and TAT-NR2B9c in theglutamate excitotoxicity model when peptides present in neuronalcultures for 15 min before and during 5-min glutamic acid exposure.Neuronal viability measured 20-24 hours following glutamic acid.Concentration of peptide in μM. MTS data were expressed as percentageneuronal viability with no insult control taken as 100% viability(mean±SD; n=4; *P<0.05).

FIG. 37 shows neuroprotective efficacy of R15 and TAT-NR2B9c in the NMDAexcitotoxicity model when peptides present in neuronal cultures for 15min before and during 5-min NMDA exposure. Neuronal viability measured20-24 hours following glutamic acid. Concentration of peptide in μM. MTSdata were expressed as percentage neuronal viability with no insultcontrol taken as 100% viability (mean±SD; n=4; *P<0.05).

FIG. 38 shows neuroprotective efficacy of R8 and Cal/R9 in the glutamateexcitotoxicity model when peptides present in neuronal cultures for 15min before and during 5-min glutamic acid exposure. Neuronal viabilitymeasured 20-24 hours following glutamic acid, Concentration of peptidein μM. MTS data were expressed as percentage neuronal viability with noinsult control taken as 100% viability (mean±SD; n=4; *P<0.05).

FIG. 39 shows neuroprotective efficacy of R9D, R12, R15 and PYC36-TATpeptides incubated with ±heparin (20 IU/ml) for 5 min at roomtemperature before for being added to neuronal cultures for 10 min onlyprior to glutamic acid exposure. Neuronal viability measured 20-24 hoursfollowing glutamic acid. Concentration of peptide in μM. MTS data wereexpressed as percentage neuronal viability with no insult control takenas 100% viability (mean±SD; n=4; *P<0.05).

FIG. 40 shows neuroprotective efficacy R9D, R12, and R15 and glutamatereceptor blockers (Blks: 5μM MK801/5μM CNQX) when neuronal culturesincubated±heparin (40 IU/ml) for 5 min at 37° C. before addition ofpeptides for 10 min only at 37° C., and then removed prior to glutamicacid exposure. Neuronal viability measured 20-24 hours followingglutamic acid. Concentration of peptide in μM. MTS data were expressedas percentage neuronal viability with no insult control taken as 100%viability (mean±SD; n=4; *P<0.05).

FIG. 41 shows neuroprotective efficacy of kFGF, kFGF-JNKI-1, TAT-JNKI-1and JNKI-1TATD in the glutamate excitotoxicity model when peptidespresent in neuronal cultures for 15 min before and during 5-min glutamicacid exposure. Neuronal viability measured 20-24 hours followingglutamic acid. Concentration of peptide in μM. MTS data were expressedas percentage neuronal viability with no insult control taken as 100%viability (mean±SD; n=4; *P<0.05).

FIG. 42 shows the results of pre-exposure of protamine sulphate (Ptm)and R18 peptides to primary rat astrocytes for 15min prior tooxygen-glucose deprivation. Concentration of peptides 2 μM. Cellviability assessed 24 hours after OGD using MTS assay. MTS data wereexpressed as absorbance values at 490 mm (mean±SD; n=4-6; *P<0.05).

FIG. 43 shows the results of exposure of protamine peptides 1-5(Ptm1-Ptm5), protamine sulphate (Ptm), and low molecular weightprotamine (LMWP) to primary rat astrocytes; concentration of protaminein μM. Treatment of astryocyte cultures with protamine was for 24 hours.Cell viability 24 hours following protamine exposure or no treatment(Control). MTS data were expressed as absorbance values at 490mm (n=4).

FIG. 44 shows intracellular calcium influx kinetics for glutamatereceptor blockers (MK801/CNQX at 5μM/5μM), R9D, R15, PYC36-TAT, TAT,TAT-NR2B9c JNKI-1-TATD, TAT-JNKI-1, kFGF-JNKI-1 and kFGF followingglutamic acid exposure in neuronal cultures. Fura-2 AM was used forintracellular calcium assessment. Representative fluorescent Fura-2 AMtracers; fluorescence intensity (FI) of neuronal cultures 30 sec beforeand following addition (arrow) of glutamic acid (100 μM finalconcentration). Peptides or glutamate receptor blockers were added toneuronal cultures for 10 min and removed (time 0) before glutamic acid.Values are mean±SE; n=3. Peptide concentration was 5μM.

FIG. 45. Diagrammatic representation of proposed model of arginine-richCPPs inducing endocytic internalization of neuronal cell surfacestructures. Note: model applies to neuronal synaptic and extra-synapticplasma membranes and potentially the plasma membrane of astrocytes,pericytes, brain endothelial cells, oligodentrocytes and microglia.NMDAR: N-methyl-D-aspartate receptors; AMPAR:a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; NCX:sodium calcium exchanger; VGCC: voltage-gated calcium channels (e.g.CaV2.2, CaV3.3); ASIC: acid-sensing ion channels; TRPM2/7: transientreceptor potential cation channels 2 and 7: mGluR: metabotropicglutamate receptors; VR1: vanilloid receptor 1 or transient receptorpotential cation channel subfamily V member 1; TNFR: tumor necrosisfactor receptors; EAAT: excitatory amino-acid transporters; AQP4:Aquaporin 4; Trk: tropomyosin-receptor-kinase receptors.

FIGS. 46 and 47 are reference tables of the sequences described and usedin this specification.

DESCRIPTION OF EMBODIMENTS

Throughout this specification, unless the context requires otherwise,the word “comprise” or variations such as “comprises” or “comprising” or“includes” or “including”, will be understood to imply the inclusion ofa stated integer or group of integers but not the exclusion of any otherinteger or group of integers.

The invention relates to isolated peptides and compositions comprisingisolated peptides, and uses thereof. The isolated peptides arecharacterized in that they can reduce the neurodegenerative effects of aneural injury or cerebrovascular ischemic event (e.g., and especially,stroke) when administered before or after the neural injury or ischemicevent. Thus, administration of the compositions of the invention reducesthe loss of neuronal cells that follows a neural injury orcerebrovascular ischemic event. The Applicant has now found,surprisingly, that certain polypeptides in the form of CPPs andpolypeptides having contiguous stretches of basic/cationic amino acids(particularly arginine residues, but also including lysine andtryptophan residues), exhibit neuroactive or neuroprotective activityand can serve as neuroprotective agents (i.e. for treatment of neuralinjury) by themselves, i.e. without having to be fused to otherneuroprotective agents or peptides. As such, the invention pertains topolypeptides of between 10 and 32 amino acids in length wherein between10 and 22 of the amino acids are cationic amino acid residues (typicallyarginine residues), with such peptides typically having an argininecontent of 30% or higher, such peptides exhibiting neuroprotectiveactivity in established neural injury models. This includespoly-arginine peptides, as well as protamine sulphate (as mixture ofprotamine peptides obtained from salmon sperm), and various versions,analogues, variants, or fragments of these peptides, including protamineand Low Molecular Weight Protamine (LMWP), and mixtures thereof.

The term “amino acid” or “residue” as used herein includes any one ofthe twenty naturally-occurring amino acids, the D-form of any one of thenaturally-occurring amino acids, non-naturally occurring amino acids,and derivatives, analogues and mimetics thereof. Any amino acid,including naturally occurring amino acids, may be purchased commerciallyor synthesized by methods known in the art. Examples ofnon-naturally-occurring amino acids include norleucine (“Nle”),norvaline (“Nva”), L- or D-naphthalanine, ornithine (“Orn”),homoarginine (homoArg) and others well known in the peptide art,including those described in M. Bodanzsky, “Principles of PeptideSynthesis,” 1st and 2nd revised ed., Springer-Verlag, New York, N.Y.,1984 and 1993, and Stewart and Young, “Solid Phase Peptide Synthesis,”2nd ed, Pierce Chemical Co., Rockford, Ill., 1984, both of which areincorporated herein by reference.

Common amino acids may be referred to by their full name, standardsingle-letter notation (IUPAC), or standard three-letter notation forexample: A, Ala, alanine; C, Cys, cysteine; D, Asp, aspartic; E, Glu,glutamic acid; F, Phe, phenylalanine; G, Gly, glycine; H, His,histidine; I, Ile isoleucine; K, Lys, lysine; L, Leu, leucine; M, Met,methionine; N, Asn, asparagine; P, Pro, proline; Q, Gln, glutamine; R,Arg, arginine; S., Ser, swine; T, Thr, threonine; V, Val, valine; W,Trp, tryptophan; X, Hyp, hydroxyproline; Y, Tyr, tyrosine. Any and allof the amino adds in the compositions herein can be naturally occurring,synthetic, and derivatives or mimetics thereof.

As used herein, “isolated” means a peptide described herein that is notin a natural state (e.g. it is disassociated from a larger proteinmolecule or cellular debris in which it naturally occurs or is normallyassociated with), or is a non-naturally occurring fragment of anaturally occurring protein (e.g. the peptide comprises less than 25%,preferably less than 10% and most preferably less than 5% of thenaturally occurring protein). Isolated also may mean that the amino acidsequence of the peptide does not occur in nature, for example, becausethe sequence is modified from a naturally occurring sequence (e.g. byalteration of certain amino acids, including basic (i.e. cationic) aminoacids such as arginine, tryptophan, or lysine), or because the sequencedoes not contain flanking amino acids which are present in nature. Theterm “isolated” may mean that the peptide or amino acid sequence is aman-made sequence or polypeptide and may be non-naturally occurring.

Likewise, “isolated” as used in connection with nucleic acids whichencode peptides embraces all of the foregoing, e.g. the isolated nucleicacids are disassociated from adjacent nucleotides with which they areassociated in nature, and can be produced recombinantly, synthetically,by purification from biological extracts, and the like. Isolated nucleicacids can contain a portion that encodes one of the foregoing peptidesand another portion that codes for another peptide or protein. Theisolated nucleic acids also can be labeled. The nucleic acids includecodons that are preferred for animal, bacterial, plant, or fungal usage.In certain embodiments, the isolated nucleic acid is a vector, such asan expression vector, which includes a nucleic acid that encodes one ofthe foregoing isolated peptides. A general method for the constructionof any desired DNA sequence is provided, e.g., in Brown J. et al.(1979), Methods in Enzymology, 68:109; Sambrook J, Maniatis T (1989),supra.

Non-peptide analogues of peptides, e.g., those that provide a stabilizedstructure or lessened biodegradation, are also contemplated. Peptidemimetic analogues can be prepared based on a selected peptide byreplacement of one or more residues by non-peptide moieties. Preferably,the non-peptide moieties permit the peptide to retain its naturalconformation, or stabilize a preferred, e.g., bioactive, conformation.One example of methods for preparation of non-peptide mimetic analoguesfrom peptides is described in Nachman et al., Regul. Pept. 57:359-370(1995). The term “peptide” as used herein embraces all of the foregoing.

As mentioned above, the peptide of the present invention may be composedeither of naturally occurring amino acids, i.e. L-amino acids, or ofD-amino acids, i.e. of an amino acid sequence comprising D-amino acidsin retro-inverso order as compared to the native sequence. The term“retro-inverso” refers to an isomer of a linear peptide in which thedirection of the sequence is reversed and the chirality of each aminoacid residue is inverted. Thus, any sequence herein, being present inL-form is also inherently disclosed herein as a D-enantiomeric(retro-inverso) peptide sequence. D-enantiomeric (retro-inverso) peptidesequences according to the invention can be constructed, e.g. bysynthesizing a reverse of the amino acid sequence for the correspondingnative L-amino acid sequence. In D-retro-inverse enantiomeric peptides,e.g. a component of the isolated peptide, the positions of carbonyl andamino groups in each single amide bond are exchanged, while the positionof the side-chain groups at each alpha carbon is preserved.

Preparation of a component of the isolated peptides of the invention asdefined above having D-enantiomeric amino acids can be achieved bychemically synthesizing a reverse amino acid sequence of thecorresponding naturally occurring L-form amino acid sequence or by anyother suitable method known to a skilled person. Alternatively, theD-retro-inverso-enantiomeric form of an peptide or a component thereofmay be prepared using chemical synthesis as disclosed above utilizing anL-form of an peptide or a component thereof as a matrix for chemicalsynthesis of the D-retro-inverso-enantiomeric form.

A cationic amino acid-rich polypeptide (which can also be referred to asa cationic amino acid polymer or copolymer) can include a polypeptide oroligomer of 10 to 32 amino acids in length. As such, by “cationic-rich”is meant any peptide, oligopeptide, or polypeptide that comprises orincludes, typically, more than 30% cationic residues, more than 50%, oreven more than 60%. In certain embodiments this may entail peptidescomprising 90%, or even 100% cationic residues such as, preferably,arginine residues. Accordingly, by an arginine-rich polypeptide (whichcan also be referred to as an arginine amino acid polymer or copolymer)can include a polypeptide or oligomer of 10 to 32 amino acids in length.As such, by “arginine-rich” is meant any peptide, oligopeptide, orpolypeptide that comprises or includes 10 or more arginine residues, ormore than 30% arginine residues, more than 50%, or even more than 60%.As such, certain embodiments comprise peptides in which 100% of theamino acids are arginine residues, with suitable efficacy and lowtoxicity when used in the range of R10 to R18 and at pharmaceuticallyefficient dosages, while in other cases it refers to other peptides(such as CPPs and including protamine and LMWP) which have intermittentstretches of arginine residues. Usually, the stretches of arginineresidues comprise consecutive/contiguous 4 to 5 arginine residues, beinginterspersed by other amino acid residues. in preferred embodiments, theinterspersed amino acids are lysine (K) residues, since these also havea generally cationic charge.

In certain embodiments, an arginine polymer or copolymer includes atleast 11 contiguous arginine residues, more preferably at least 12contiguous arginine residues, more preferably at least 13 contiguousarginine residues, more preferably at least 14 contiguous arginineresidues, more preferably at least 15 contiguous arginine residues, morepreferably at least 16 contiguous arginine residues, more preferably atleast 17 contiguous arginine residues and more preferably at least 18contiguous arginine residues. However, in certain embodiments, there maybe contiguous sequences of 4 to 5 arginine residues interspersed byother, non-arginine residues, such as those exemplified by protamine,LMWP, and functional variants thereof having neuroprotective activity orfor use in treating neural injury, as shown herein. In a preferredembodiment, use is made of R15.

The contiguous arginine residues can be at the C-terminus of thepolypeptide, N-terminus of the polypeptide, in the centre of thepolypeptide (e.g., surrounded by non-arginine amino acid residues), orin any position within a polypeptide. Non-arginine residues arepreferably amino acids, amino acid derivatives, or amino acid mimeticsthat do not significantly reduce the rate of membrane transport of thepolymer into cells, including, for example, glycine, alanine, cysteine,valine, leucine, isoleucine, methionine, serine, threonine,α-amino-beta-guanidinopropionic acid, α-amino-γ-guanidinobutyric acid,and α-amino-ϵ-guanidinocaproic acid.

Various changes may be made including the addition of various sidegroups that do not affect the manner in which the peptide functions, orwhich favourably affect the manner in which the peptide functions. Suchchanges may involve adding or subtracting charge groups, substitutingamino acids, adding lipophilic moieties that do not effect binding butthat affect the overall charge characteristics of the moleculefacilitating delivery across the blood-brain barrier, etc. For each suchchange, no more than routine experimentation is required to test whetherthe molecule functions according to the invention One simply makes thedesired change or selects the desired peptide and applies it in afashion as described in detail in the examples. For example, if thepeptide (modified or unmodified) is active in a test of protectionagainst kainic acid, or if such a peptide competes with the parentneurotransmitter in a test of neurotransmitter function, then thepeptide is a functional neurotransmitter peptide.

The invention also embraces functional variants of the isolated peptide.As used herein, a “functional variant” or “variant” of an isolatedpeptide is a peptide which contains one or more modifications to theprimary amino acid sequence of the isolated peptide and retains theproperties disclosed herein. Modifications which create a functionalvariant of the isolated peptide can be made, for example, 1) to enhancea property of an isolated peptide, such as peptide stability in anexpression system; 2) to provide a novel activity or property to anisolated peptide, such as addition of an antigenic epitope or additionof a detectable moiety; or 3) to provide a different amino acid sequencethat produces the same or similar peptide properties. Modifications toan isolated peptide can be made to a nucleic acid that encodes thepeptide, and can include deletions, point mutations, truncations, aminoacid substitutions and additions of amino acids. Alternatively,modifications can be made directly to the peptide, such as by cleavage,addition of a linker molecule, preferably a cleavable linker such asMMP, calpain, tPA, addition of a detectable moiety such as biotin,addition of a fatty acid, substitution of one amino acid for another andthe like. Modifications also embrace fusion proteins comprising all orpart of the isolated peptide amino acid sequence. In one embodiment, thelinker is selected from one or more MMP-type linkers, calpain, caspase,or tPA linkers. MMP-type linkers are defined as the peptide sequencerecognized and cleaved by matrix metalloproteinases (MMPs). Similarly acalpain, caspase or PA linker is a peptide sequence recognized andcleaved by these protease enzymes. In certain embodiments, the peptidesof the invention are also fused to other peptides that are to betransported to the site of a neural injury or are to be transportedintracellularly in neural cells. As such, the peptides of the inventionmay also be linked to ancillary peptides that can bind to or interactwith enzymes detrimental to neural function, so that the ancillarypeptides can function as competitive inhibitors of the enzymes followingtransportation across the cell membrane by the peptide of the invention.The ancillary peptides are tPA, calpain, and MMP, and are linked to thepeptide of the invention using a cleavable linker such as a caspasesequence, so that the tPA, calpain or MMP is liberated from the peptideof the invention to then function as a competitive inhibitorintracellularly for enzymes detrimental to neural function.

As such, the invention extends to a peptide of the invention, such asR10-R18 or Ptm1-5 or LMWP, or variants thereof, linked to a caspasecleavage site, itself in turn linked to calpain, tPA or MMP.

The term “sequence identity” as defined herein means that the sequencesare compared as follows. To determine the percent identity of two aminoacid sequences, the sequences can be aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid sequence). The amino acids at corresponding amino acid positionscan then be compared. When a position in the first sequence is occupiedby the same amino acid as the corresponding position in the secondsequence, then the molecules are identical at that position. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences. For example, where aparticular peptide is said to have a specific percent identity to areference polypeptide of a defined length, the percent identity isrelative to the reference peptide. Thus, a peptide that is 50% identicalto a reference polypeptide that is 100 amino acids long can be a 50amino acid polypeptide that is completely identical to a 50 amino acidlong portion of the reference polypeptide. It might also be a 100 aminoacid long polypeptide, which is 50% identical to the referencepolypeptide over its entire length. Such a determination of percentidentity of two sequences can be accomplished using a mathematicalalgorithm. A preferred, non-limiting example of a mathematical algorithmutilized for the comparison of two sequences is the algorithm of Karlinet al. (1993), PNAS USA, 90:5873-5877. Such an algorithm is incorporatedinto the NBLAST program, which can be used to identify sequences havingthe desired identity to the amino acid sequence of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997), Nucleic Acids Res,25:3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., NBLAST) can beused. The sequences further may be aligned using Version 9 of theGenetic Computing Group's GAP (global alignment program), using thedefault (BLOSUM62) matrix (values −4 to +11) with a gap open penalty of-12 (for the first null of a gap) and a gap extension penalty of −4 (pereach additional consecutive null in the gap). After alignment,percentage identity is calculated by expressing the number of matches asa percentage of the number of amino acids in the claimed sequence. Thedescribed methods of determination of the percent identity of two aminoacid sequences can be applied correspondingly to nucleic acid sequences.

The peptide of the invention may be linked directly or via a linker. A“linker” in the present context is usually a peptide, oligopeptide orpolypeptide and may be used to link multiples of the peptides to oneanother. The peptides of the invention selected to be linked to oneanother can be identical sequences, or are selected from any of thepeptides of the invention. A linker can have a length of 1-10 aminoacids, more preferably a length of 1 to 5 amino acids and mostpreferably a length of 1 to 3 amino acids. In certain embodiments, thelinker is not required to have any secondary structure formingproperties, i.e. does not require a α-helix or β-sheet structure formingtendency, e.g. if the linker is composed of at least 35% of glycineresidues. As mentioned hereinbefore, a linker can be a cleavable peptidesuch as an MMP peptide which can be cleaved intracellularly by normalcellular processes, effective raising the intracellular dose of thepreviously linked peptides, while keeping the extracellular dose lowenough to not be considered toxic. The use of a(n)intracellularly/endogenously cleavable peptide, oligopeptide, orpolypeptide sequence as a linker permits the peptides to separate fromone another after delivery into the target cell. Cleavable oligo- orpolypeptide sequences in this context also include protease cleavableoligo- or polypeptide sequences, wherein the protease cleavage site istypically selected dependent on the protease endogenously expressed bythe treated cell. The linker as defined above, if present as an oligo-or polypeptide sequence, can be composed either of D-amino acids or ofnaturally occurring amino acids, i.e. L-amino acids. As an alternativeto the above, coupling or fusion of the peptides can be accomplished viaa coupling or conjugating agent, e.g a cross-linking reagent. There areseveral intermolecular cross-linking reagents which can be utilized, seefor example, Means and Feeney, Chemical Modification of Proteins,Holden-Day, 1974, pp. 39-43. Among these reagents are, for example,N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) orN,N′-(1,3-phenylene)bismaleimide; N,N′-ethylene-bis-(iodoacetamide) orother such reagent having 6 to 11 carbon methylene bridges; and1,5-difluoro-2,4-dinitrobenzene. Other cross-linking reagents useful forthis purpose include: p,p′-difiuoro-m,m′-dinitrodiphenylsulfone;dimethyl adipimidate; phenol-1,4-disulfonylchloride;hexamethylenediisocyanate or diisothiocyanate, orazophenyl-p-diisocyanate; glutaraldehyde and disdiazobenzidine.Cross-linking reagents may be homobifunctional, i.e., having twofunctional groups that undergo the same reaction. A preferredhomobifunctional cross-linking reagent is bismaleimidohexane (BMH). BMHcontains two maleimide functional groups, which react specifically withsulfhydryl-containing compounds under mild conditions (pH 6.5-7.7). Thetwo maleimide groups are connected by a hydrocarbon chain. Therefore,BMH is useful for irreversible cross-linking of proteins (orpolypeptides) that contain cysteine residues. Cross-linking reagents mayalso be heterobifunctional. Heterobifunctional cross-linking reagentshave two different functional groups, for example an amine-reactivegroup and a thiol-reactive group, that will cross-link two proteinshaving free amines and thiols, respectively, Examples ofheterobifunctional cross-linking reagents are succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and succinimide4-(p-maleimidophenyl)butyrate (SMPB), an extended chain analogue of MBS,The succinimidyl group of these cross-linking reagents with a primaryamine, and the thiol-reactive maleimide forms a covalent bond with thethiol of a cysteine residue. Because cross-linking reagents often havelow solubility in water, a hydrophilic moiety, such as a sulfonategroup, may be added to the cross-linking reagent to improve its watersolubility. Sulfo-MBS and sulfo-SMCC are examples of cross-linkingreagents modified for water solubility. Many cross-linking reagentsyield a conjugate that is essentially non-cleavable under cellularconditions. Therefore, some cross-linking reagents contain a covalentbond, such as a disulfide, that is cleavable under cellular conditions.For example, Traut's reagent, dithiobis (succinimidylpropionate) (DSP),and N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) are well-knowncleavable cross-linkers. The use of a cleavable cross-linking reagentpermits the peptides to be separated after delivery into the targetcell, if desired, provided the cell is capable of cleaving a particularsequence of the crosslinker reagent. For this purpose, direct disulfidelinkage may also be useful. Chemical cross-linking may also include theuse of spacer arms. Spacer arms provide intramolecular flexibility oradjust intramolecular distances between conjugated moieties and therebymay help preserve biological activity. A spacer arm may be in the formof a protein (or polypeptide) moiety that includes spacer amino acids,e.g. praline. Alternatively, a spacer arm may be part of thecross-linking reagent, such as in “long-chain SPDP” (Pierce Chem. Co.,Rockford, Ill., cat. No. 21651H). Numerous cross-linking reagents,including the ones discussed above, are commercially available. Detailedinstructions for their use are readily available from the commercialsuppliers. A general reference on protein cross-linking and conjugatepreparation is: Wong, Chemistry of Protein Conjugation andCross-Linking, CRC Press (1991).

The peptides of the invention may also contain a “derivative”,“variant”, or “functional fragment”, i.e. a sequence of a peptide thatis derived from the naturally occurring (L-amino-acid) sequence of apeptide of the invention as defined above by way of substitution(s) ofone or more amino acids at one or more of sites of the amino acidsequence, by way of deletion(s) of one or more amino acids at any siteof the naturally occurring sequence, and/or by way of insertion(s) ofone or more amino acids at one or more sites of the naturally occurringpeptide sequence. “Derivatives” shall retain their biological activityif used as peptides of the invention, e.g. a derivative of any of thepeptides of the invention shall retain its neuroprotective activity.Derivatives in the context of the present invention may also occur inthe form of their L- or D-amino-acid sequences as defined above, orboth.

If substitution(s) of amino acid(s) are carried out for the preparationof a derivative of the peptides of the invention, conservative (aminoacid) substitutions are preferred. Conservative (amino acid)substitutions typically include substitutions within the followinggroups: glycine and alanine; valine, isoleucine and leucine; asparticacid and glutamic acid; asparagine and glutamine; serine and threonine;lysine and arginine; and phenylalanine and tyrosine. Thus, preferredconservative substitution groups are aspartate-glutamate;asparagine-glutamine; valine-leucine-isoleucine; alanine-valine; andphenylalanine-tyrosine. By such mutations e.g. stability and/oreffectiveness of a peptide may be enhanced. If mutations are introducedinto the peptide, the peptide remains (functionally) homologous, e.g. insequence, in function, and in antigenic character or other function.Such mutated components of the peptide can possess altered propertiesthat may be advantageous over the non-altered sequences of the peptidesof the invention for certain applications (e.g. increased pH optimum,increased temperature stability etc.).

A derivative of the peptide of the invention is defined as havingsubstantial identity with the non-modified sequences of the peptide ofthe invention. Particularly preferred are amino acid sequences whichhave at least 30% sequence identity, preferably at least 50% sequenceidentity, even preferably at least 60% sequence identity, evenpreferably at least 75% sequence identity, even more preferably at least80%, yet more preferably 90% sequence identity and most preferably atleast 95% or even 99% sequence identity to the naturally occurringanalogue. Appropriate methods for synthesis or isolation of a functionalderivative of the peptides of the invention as well as for determinationof percent identity of two amino acid sequences are described above.Additionally, methods for production of derivatives of the peptides asdisclosed above are well known and can be carried out following standardmethods which are well known by a person skilled in the art (see e.g.,Sambrook J, Maniatis T (1989)).

As a further embodiment, the invention provides pharmaceuticalcompositions or medicaments comprising the peptides as defined herein.In certain embodiments, such pharmaceutical compositions or medicamentscomprise the peptides as well as an optional linker, as defined herein.Additionally, such a pharmaceutical composition or medicament cancomprise a pharmaceutically acceptable carrier, adjuvant, or vehicle. A“pharmaceutically acceptable carder, adjuvant, or vehicle” according tothe invention refers to a non-toxic carrier, adjuvant or vehicle thatdoes not destroy the pharmacological activity or physiological targetingof the peptide with which it is formulated. Pharmaceutically acceptablecarriers, adjuvants or vehicles that can be used in the pharmaceuticalcompositions of this invention include, but are not limited to thosethat can be applied cranially or intracranially, or that can cross theblood-brain barrier (BBB). Notwithstanding this, the pharmaceuticalcompositions of the invention can include ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The pharmaceutical compositions of the present invention may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally, cerebrally, or via an implantedreservoir.

The term parenteral as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. The pharmaceutical compositions are administeredorally, intraperitoneally or intravenously. Sterile injectable forms ofthe pharmaceutical compositions of this invention may be aqueous oroleaginous suspension. These suspensions can be formulated according totechniques known in the art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium.

As such, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents that arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation.

The pharmaceutically acceptable compositions herein may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents, If desired, certain sweetening,flavouring or colouring agents may also be added.

Alternatively, the pharmaceutical composition as defined herein may beadministered in the form of suppository for rectal administration. Sucha suppository can be prepared by mixing the agent with a suitablenon-irritating excipient that is solid at room temperature but liquid atrectal temperature and, therefore, will melt in the rectum to releasethe drug. Such materials include cocoa butter, beeswax and polyethyleneglycols.

The pharmaceutical composition as defined herein may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the brain, other intra-cranial tissues, the eye, or theskin. Suitable formulations are readily prepared for each of these areasor organs.

For topical applications, the pharmaceutical composition as definedherein may be formulated in a suitable ointment containing the peptidesas identified herein, suspended or dissolved in one or more carriers.Carriers for topical administration of the peptide include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical composition as defined hereincan be formulated in a suitable lotion or cream containing the peptidesuspended or dissolved in one or more pharmaceutically acceptablecarriers Suitable carriers include, but are not limited to, mineral oil,sorbitan moNOstearate, polysorbate 60, cetyl esters wax, cetearylalcohol, 2-octyldodecanol, benzyl alcohol and water.

The pharmaceutical composition as defined herein may also beadministered by nasal aerosol or inhalation. Such a composition may beprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents, The pharmaceutically acceptable composition ormedicament herein is formulated for oral or parenteral administration,e.g. by injection.

For treatment purposes, a non-toxic, damage-reducing, effective amountof the peptide may be used for preparation of a pharmaceuticalcomposition as defined above. Therefore, an amount of the peptide may becombined with the carrier material(s) to produce a composition asdefined above. The pharmaceutical composition is typically prepared in asingle (or multiple) dosage form, which will vary depending upon thehost treated and the particular mode of administration. Usually, thepharmaceutical composition is formulated so that a dosage range per doseof 0.0001 to 100 mg/kg body weight/day of the peptide can beadministered to a patient receiving the pharmaceutical composition.Preferred dosage ranges per dose vary from 0.01 mg/kg body weight/day to50 mg/kg body weight/day, even further preferred dosage ranges per doserange from 0.1 mg/kg body weight/day to 10 mg/kg body weight/day.However, dosage ranges and treatment regimens as mentioned above may beadapted suitably for any particular patient dependent upon a variety offactors, including the activity of the specific peptide employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, the judgment of the treatingphysician and the severity of the particular disease being treated. Inthis context, administration may be carried with in an initial dosagerange, which may be varied over the time of treatment, e.g. byincreasing or decreasing the initial dosage range within the range asset forth above. Alternatively, administration may be carried out in acontinuous manner by administering a specific dosage range, therebymaintaining the initial dosage range over the entire time of treatment.Both administration forms may furthermore be combined, e.g. if thedosage range is to be adapted (increased or decreased) between varioussessions of the treatment but kept constant within the single session sothat dosage ranges of the various sessions differ from each other.

The pharmaceutical composition and/or the peptide of the invention canbe used for treatment, amelioration or prevention of diseases related tothe damaging effect of an injury to cells, particularly mammalian cells,as disclosed herein, particularly for the treatment of neural injuries,including cerebral stroke or spinal cord injuries, epilepsy, perinatalhypoxia-ischemia, ischemic or traumatic injuries to the brain or spinalcord and damages to central nervous system (CNS) neurons including,without being limited thereto, acute CNS injuries, ischemic cerebralstroke or spinal cord injuries, as well as of anoxia, ischemia,mechanical injury, neuropathic pain, excitotoxicity, and relatedinjuries. Furthermore, the pharmaceutical composition and peptides ofthe invention can be employed for providing a neuroactive orneuroprotective effect against, or treatment of, excitotoxic andischemic injury, excitotoxicity, lack of neurotrophic support,disconnection, damage to neurons including e.g. epilepsy, chronicneurodegenerative conditions, and the like. In this context,excitotoxicity may be particularly involved in stroke, traumatic braininjury and neurodegenerative diseases of the central nervous system(CNS) such as Multiple sclerosis (MS), Alzheimer's disease (AD),Amyotrophic lateral sclerosis (ALS), neuropathic pain, Fibromyalgia,Parkinson's disease (PD), perinatal hypoxia-ischemia, and Huntington'sdisease, that can be treated herein. Other common conditions that causeexcessive glutamate concentrations around neurons and which may betreated herein are hypoglycemia, vasospasm, benzodiazepine withdrawaland status epilepticus, glaucoma/deterioration of retinal ganglioncells, and the like.

The treatment, amelioration or prevention of diseases related to thedamaging effect of an injury to mammalian cells as defined above as wellas to further diseases or disorders as mentioned herein is typicallycarried out by administering a pharmaceutical composition or peptide ormixture of peptides of the invention in a dosage range as describedherein. Administration of the pharmaceutical composition or peptides maybe carried out either prior to onset of excitotoxicity and/or (ischemic)brain damage, i.e. the damaging effect of an injury to mammalian cells,or concurrent or subsequent thereto; for example, administration of thepharmaceutical composition or peptides may be carried out within a timeof (up to) 1 hour (0-1 hours), up to 2 hours, up to 3-5 hours or up to24 hours or more subsequent to a cerebral stroke or spinal cordinjuries, ischemic or traumatic injuries to the brain or spinal cordand, in general, damages to the central nervous system (CNS) neurons. Inchronic neurodegenerative disorders (AD, PD, ALS, MS, etc.) treatmentmay require life-long daily treatment.

When used therapeutically, the compounds of the invention areadministered in therapeutically effective amounts. In general, atherapeutically effective amount means an amount necessary to delay theonset of, inhibit the progression of, or halt altogether the particularcondition being treated. Therapeutically effective amounts specificallywill be those that desirably influence the survival of neurons followingstroke or other cerebral ischemic insult. Generally, a therapeuticallyeffective amount will vary with the subject's age and condition, as wellas the nature and extent of the disease in the subject, all of which canbe determined by one of ordinary skill in the art. The dosage may beadjusted by the individual physician, particularly in the event of anycomplications being experienced.

As mentioned above, one aspect of the invention relates to nucleic acidsequences and their derivatives which code for an isolated peptide orvariant thereof and other nucleic acid sequences which hybridize to anucleic acid molecule consisting of the above described nucleotidesequences, under stringent conditions. The term “stringent conditions”as used herein refers to parameters with which the art is familiar.Nucleic acid hybridization parameters may be found in references whichcompile such methods, e.g. Molecular Cloning: A Laboratory Manual, J.Sambrook, et al., eds., Second Edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, or Current Protocols in MolecularBiology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.More specifically, stringent conditions, as used herein, refers tohybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll,0.02% Polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 25 mM NaH₂PO₄(pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M Sodium Chloride/0.15 M SodiumCitrate, pH 7; SDS is Sodium Dodecyl Sulphate; and EDTA is Ethylenediaminetetraacetic acid. After hybridization, the membrane upon whichthe DNA is transferred is washed at 2×SSC at room temperature and thenat 0.1×SSC/0.1×SDS at 65° C.

The present invention furthermore provides kits comprising theabovementioned pharmaceutical composition (in one or more containers) inat least one of the above formulations and an instruction manual orinformation brochure regarding instructions and/or information withrespect to application of the pharmaceutical composition.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

Those skilled in the field of the invention will appreciate that theinvention described herein is susceptible to variations andmodifications other than those specifically described. It is to beunderstood that the invention includes all such functional variationsand modifications. The invention also includes all of the steps,features, compositions and compounds referred to or indicated in thisspecification, individually or collectively, and any and allcombinations or any two or more of said steps or features. The presentinvention is not to be limited in scope by the specific embodimentsdescribed herein, which are intended for the purpose of exemplificationonly. Functionally-equivalent products, compositions and methods areclearly within the scope of the invention, as described herein.Furthermore, the present invention is performed without undueexperimentation using, unless otherwise indicated, conventionaltechniques of molecular biology, microbiology, neurobiology, virology,recombinant DNA technology, peptide synthesis in solution, solid phasepeptide synthesis, and immunology, or techniques cited herein.

The Applicant has found, surprisingly, that certain CPPs, especiallyarginine-rich peptides exhibit neuroprotective efficacy without thetraditional requirement for being fused to previously identifiedneuroprotective peptides. Certain of these CPPs also exhibit lowtoxicity and are functional at low doses or concentrations. These CPPsinclude penetratin and Pep-1. More surprisingly, however, the Applicanthas also found that long stretches of basic (i.e. cationic) amino acidssuch as polyarginine peptides of between 10 and 20 residues (inclusive)in length, preferably 10 to 18 residues (inclusive) in length, andcertain peptides containing more than 10 arginine residues, such asprotamine or LMWP, exhibit greatly enhanced neuroprotective activitywhen compared to these CPPs, and especially when compared at similarconcentrations to shorter arginine-rich sequences such as R1 to R8. Inparticular, R12, R15, R18, when assayed in glutamic acid and or in vitroischemia injury models, provided enhanced neuroprotection when comparedto the CPPs mentioned above. The two different neuronal injury modelsare likely to activate different damaging cellular pathways and therebywill provide further insight into the neuroprotective spectrum andpossible mode of action of the peptides of the invention.

In this specification, the abbreviation “Arg” followed by an integerindicates the number of arginine repeats in a peptide. Thus, Arg-15(abbreviated as R15, following the IUPAC single letter abbreviation forarginine) refers to consecutive arginine residues in a peptideformation. As far as practicable, however, this specification will referto the single letter amino acid code, i.e. R15, instead of Arg-15, forexample.

Neuronal Disorders Involving Neuronal Cell Death

Neuronal disorders such as migraine, stroke, traumatic brain injury,spinal cord injury epilepsy, perinatal hypoxia-ischemia andneurodegenerative disorders including Huntington's Disease (HD),Parkinson's Disease (PD), Alzheimer's Disease (AD) and AmyotrophicLateral Sclerosis (ALS) are major causes of morbidity and disabilityarising from long term brain injury. The brain injuries generallyinvolve a range of cell death processes including apoptosis, autophagy,necroptosis and necrosis, and affect neurons astrocytes,oligodentrocytes, microglia and vascular endothelial cells (collectivelyreferred to as the neurovascular unit; NVU). The damaging triggersinvolved in neural injury involve diverse pathways involving glutamateexcitotoxicity calcium overload, oxidative stress, proteolytic enzymesand mitochondrial disturbances. As used herein, the term “stroke”includes any ischemic disorder affecting the brain or spinal cord, e.g.thrombo-embolic occlusion in a brain or spinal cord artery, severehypotension, perinatal hypoxia-ischaemia, a myocardial infarction,hypoxia, cerebral haemorrhage, vasospasm, a peripheral vasculardisorder, a venous thrombosis, a pulmonary embolus, a transientischaemic attack, lung ischemia, unstable angina, a reversible ischemicneurological deficit, adjunct thrombolytic activity, excessive clottingconditions, cerebral reperfusion injury, sickle cell anemia, a strokedisorder or an iatrogenically induced ischemic period such asangioplasty, or cerebral ischemia.

Increased extracellular levels of the neurotransmitter glutamate cancause neuronal cell death via acute and delayed damaging processedcaused by excitotoxicity. An accumulation of extracellular glutamateover-stimulates NMDA and AMPA receptors and subsequently, VGCCs, NCX,TRMP2/7, ASIC and mGlu receptors resulting in an influx of extracellularcalcium and sodium ions and the release of bound calcium fromintracellular stores. Over-activation of NMDA receptors can also triggerthe production of damaging molecules (e.g. nitric oxide, CLCA1;calcium-activated chloride channel regulator 1, calpain, SREBPI: sterolregulatory element binding protein-1) and signaling pathways (e.g. DAPK;death-associated protein kinase, CamKII: calcium-calmodulin-dependentprotein kinase II). The increase in intracellular calcium initiates arange of cell damaging events involving phospholipases, proteases,phosphatases, kinases and nitric oxide synthase, as well as theactivation of pathways triggering cell death (i.e. apoptosis, autophagy,necroptosis and necrosis).

Since the peptides of the invention are shown herein to protect neuronsfrom death, the disclosures contained in WO2009133247 and EP 1969003show that the peptides of the invention also find application in thetreatment and/or prevention of Alzheimer's disease, Parkinson's disease,amyotrophic lateral sclerosis, stroke, peripheral neuropathy, orepilepsy and accordingly to other associated pathologies describedherein. Accordingly, the present invention is directed to a method fortreatment of Alzheimers disease, Parkinson's disease, amyotrophiclateral sclerosis, stroke, peripheral neuropathy, epilepsy, spinal cordinjury, diabetes or drug addiction, wherein a pharmaceutically effectiveamount of any one or more of the peptides of the invention isadministered to a patient. In other words, the peptides according to thepresent invention are for use in the treatment of injuries associatedwith ischemia, as well as Alzheimer's disease (AD), Huntington's disease(HD), Parkinson's disease (PD), multiple sclerosis (MS), acutedisseminated encephalomyelitis (ADEM), amyotrophic lateral sclerosis(ALS), stroke, peripheral neuropathy, epilepsy, spinal cord injury andother related pathologies described herein.

In pharmaceutical applications, the peptides can also be entrapped inmicrocapsules prepared, for example, by co-acervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences. This may also be accomplished usingsustained-release preparations. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymers containing the peptide, which matrices are in the form ofshaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels as described byLanger et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer,Chem. Tech., 12:98-105 (1982) or polyvinylalcohol, polylactides (U.S.Pat. No. 3,773,919, EP 58,481), or non-degradable ethylene-vinylacetate.

In one embodiment, a pharmaceutical composition comprising the peptideof the invention as defined above is for use in the treatment ofischemic injury, Alzheimer's disease, Parkinson's disease, amyotrophiclateral sclerosis, stroke, peripheral neuropathy, or epilepsy. Inanother embodiment, the present invention provides a method forpromoting survival of neurons comprising the step of contacting neuronswith the peptides of the invention, or combinations thereof. The methodcan be performed in vitro as is shown herein.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, to provide additionaldetails with respect to its practice, are incorporated herein byreference. The present invention is further described in the followingexamples, which are not intended to limit the scope of the invention.

EXAMPLES (Poly-arginine and Arginine-rich and Arginine-rich ProtaminePeptides of Tables 1, 2 and 5 below) Protamine Sulphate, ProtaminePeptides and Other Peptides

Peptides listed in Table 5. Protamine sulphate (protamine; Ptm) wasobtained from Sanofi Aventis. Low molecular weight protamine (LMWP) wassynthesised by Mimotopes Pty Ltd (Australia). Protamine peptides 1-5(Ptm1, Ptm2, Ptm3, Ptm4, Ptm5) were synthesised by Pepmic Co Ltd(China). The peptides were HPLC purified to greater than 90-98%. Allpeptides were prepared as 100× stocks (500 μM) in normal saline andassessed in a concentration range from 0.1-10 μM, dependent upon injurymodel.

It should be noted that protamine sulphate (protamine; Ptm) is a mixtureof Ptm1-Ptm4¹. The protamine peptides (Ptm, Ptm1-5, LMWP) are argininerich.

Methods (Poly-arginine and Arginine-rich Peptides) Primary NeuronalCortical Cultures

Establishment of cortical cultures was as previously described (Meloniet al. 2001). Briefly, cortical tissue from E18-E19 Sprague-Dawley ratswas dissociated in Dulbecco's modified Eagle medium (DMEM; Invitrogen,Australia) supplemented with 1.3 mM L-cysteine, 0.9 mM NaHCO₃, 10units/ml papain (Sigma, USA) and 50 units/ml DNase (Sigma) and washed incold DMEM/10% horse serum. Neurons were resuspended in Neurobasal(Invitrogen) containing 2% B27 supplement (B27; Invitrogen). Beforeseeding, 96-well sized glass wells (6 mm diameter, ProTech, Australia)or 96-well plastic plates (ProSciTech, Australia) were coated withpoly-D-lysine overnight (50 ml/well: 50 mg/mL; 70-150 K, Sigma). Excesspoly-D-lysine solution was then removed and replaced with Neurobasal(containing 2% B27; 4% fetal bovine serum; 1% horse serum; 62.5 mMglutamate; 25 mM 2-mercaptoethanol; and 30 mg/mL streptomycin and 30mg/mL penicillin). Neurons were plated to obtain ≈10,000 viable neuronsfor each well on day in vitro 11-12. Neuronal cultures were maintainedin a CO₂ incubator (5% CO₂, 95% air balance, 98% humidity) at 37° C. Onday in vitro 4 one third of the culture medium was removed and replacedwith fresh Neurobasal/2% 827 containing the mitotic inhibitor, cytosinearabinofuraNOside (1 mM final concentration; Sigma). On day in vitro 8one half of the culture medium was replaced with Neurobasal/2′% B27.Cultures were used on day in vitro 11 or 12 after which time theyroutinely consist of >97% neurons and 1-3% astrocytes (Meloni et al.2001).

Other Cell Lines Used

A brain endothelial cell line (bEND3) and a neuroblastoma cell line(SH-SY5Y) were also used for some experiments. bEND3 and SH-SY5Y cellswere cultured using standard techniques in DMEM plus 5 or 15% foetalcalf serum. Rat primary astrocytes were obtained and cultured asdescribed for cortical neurons except DMEM plus 10% foetal calf serumwas used instead of Neurobasal/2% B27, and the mitotic inhibitor,cytosine arabinofuraNOside was not used.

Cell Penetrating Peptides And Control Peptides

Peptides listed in Table 1 were synthesised by Mimotopes Ply Ltd(Australia), except TAT-L, which was synthesised by Pepscan Presto (TheNetherlands), and R9/tPA/R9, NCXBP3 and R9/X7/R9, which were synthesisedby China Peptide Co., Ltd. Peptides listed in Table 2 were synthesisedby China Peptides (China). The peptides were HPLC purified to greaterthan 88-96%. TAT-L, penetratin, R9 and Pep-1 were synthesised in theL-isoform and TAT-D and Arg9-D in the protease resistant D-retro-inversoform, synthesised from D-amino acids in reverse sequence (referred to asD-isoform hereafter) (Brugidou et al. 1995) (Table 1). A TAT-fused MKinhibitory peptide (JNKI-1D-TAT) in the D-isoform and a TAT-fused AP-1inhibitory peptide (PYC36L-TAT) in the L-isoform were used as positivecontrols (Table 1; Borsello et al. 2003; Meade et al. 2010b). Allpeptides were prepared as 100× stocks (500 μM) in normal saline andassessed in a concentration range from 0.1-15 μM, dependent upon injurymodel. The TAT-L peptide was only used in the glutamic acidexcitotoxicity model.

TABLE 1 Amino acid sequences, molecular weights and charge of peptidesNo. amino acids/No. arginine residues: Net Physical- SEQ molecularcharge chemical Peptide ID NO. Sequence weight (Da) at pH 7 properitesR1: Arg-1  1 H-R-OH 1/1: 174 1 Cationic R3: Arg-3  2 H-RRR-OH 3/3: 487 3Cationic R6: Arg-6  3 H-RRRRRR-OH 6/6: 955 6 Cationic R7: Arg-7  4H-RRRRRRR-OH 7/7: 1,111 7 Cationic R8: Arg-8  5 H-RRRRRRRR-OH 8/8: 1,2678 Cationic R9: Arg-9  6 H-RRRRRRRRR-OH 9/9: 1,424 9 Cationic R10: Arg-10 7 H-RRRRRRRRRR-OH 10/10: 1,580 10 Cationic R11: Arg-11  8H-RRRRRRRRRRR-OH 11/11: 1,736 11 Cationic R12: Arg-12  9H-RRRRRRRRRRRR-OH 12/12: 1,892 12 Cationic R13: Arg-13 10H-RRRRRRRRRRRRR-OH 13/13: 2,048 13 Cationic R14: Arg-14 11H-RRRRRRRRRRRRRR-OH 14/14: 2,204 14 Cationic R15: Arg-15 12H-RRRRRRRRRRRRRRR-OH 15/15: 2,360 15 Cationic R18: Arg-18 13H-RRRRRRRRRRRRRRRRRR-OH 18/18: 2,829 18 Cationic PTD-4^(a) 14H-YARAAARQARA-OH 11/3: 1,204 3 Cationic E9/R9 15H-EEEEEEEEE-RRRRRRRRR-OH 18/9: 2,586 0 Neutral (R9/E9) R9/tPA/R9 16H-RRRRRRRRR-PGRVVGG-RRRRRRRRR-OH 25/19: 3,452 19 Cationic or R9/X7/R9DAHK 17 H-DAHK-OH 4/0: 469.5 0.1 N/A NR2B9c-TAT^(b) 18H-GRKKRRQRRR-KLSSIESDV-NH2 19/6: 2,355 Cationic D-R9 19 H-rrrrrrrrr-NH29/9: 1423.73 10 Cationic TAT-D 20 H-GrrrqrrkkrG-NH2 10/6: 1,453 9Cationic TAT-L 21 Ac-GRKKRRQRRRG-NH2 10/6: 1,494 8 Cationic Penetratin22 H-RQIKIWFQNRRMKWKK-NH2 16/3: 2,246 7 Cationic Pep-1 23H-KETWWETWWTEWSQPKKKRKV-NH2 21/1: 2,847 4 Amphiphilic PYC36L-TAT^(c) 24H-GRKKRRQRRR-GGLQGRRRQGYQSIKP-NH2 26/10: 3,180 13 CationicJNK1-1D-TAT^(d) 25 H-tdqsrpvqpflnlttprkprpp-rrrqrrkk 32/: 3,925 12Cationic rg-NH2 TAT-JNKI-1^(d) 26 H-GRKKRRQRRR-PPRPKRPTTLNLFPQVPRSQ32/9: 3,924.6 11 Cationic DT-OH kFGF-JNKI-1 27H-AAVALLPAVLLALLAP-PPRPKRPTTLNLFP 38/3: 4,043.9 3 HydrophobicQVPRSQDT-OH kFGF 28 H-AAVALLPAVLLALLAP-OH 16/0: 1515.96 0 HydrophobicXIP^(e) 29 H-RRLLFYKYVYKRYRAGKQRG-OH 20/5: 2621.14 8 Cationic NCXBP3^(f)30 H-RRERRRRSCAGCSRARGSCRSCRR-NH2 24/11: 2881.34 10.8 Cationic Cal/R9 31Ac-PLFAE-RRRRRRRRR-NH2 15/9: 2022.41 8 Cationic At the N-terminus, Hindicates free amine, and Ac indicates acetyl. At the C-terminus OHindicates free acid and NH2 indicates amide. AA = amino acids. Lowercase single letter code indicates D-isoform of the amino acid.^(a)Peptide describe in Ho et al (2001), ^(b)NR2B9c-TAT also shown asNA-1 (Aarts et al., 2002; Hill et al 2012), ^(c)peptide described inMeade et al. 2010ab and isolated by Phylogical Ltd, ^(d)peptidedescirbed in Borsello et al. 2003, ^(e)XIP described by He et al., 1997,^(f)peptide isolated by Jane Cross/Bruno Meloni from phylomer library(Phylogica Pty Ltd).

Glutamic Acid and Kainic Acid and NMDA Excitotoxicity Models and PeptideIncubation

Peptides were added to culture wells (96-well plate format) 15 minutesprior to glutamic acid or kainic acid exposure by removing media andadding 50 μl of Neurobasal/2% B27 containing CPPs, control peptides orMK801/CNQX. To induce excitotoxicity, 50 μl of Neurobasal/2% B27containing glutamic acid (200 μM) or kainic acid (400 μM) or NMDA (200μM) was added to the culture wells (100 μM glutamic acid, 200 μM kainicacid and NMDA 100μM final concentration). Cultures were incubated at 37°C. in the CO₂ incubator for 5 minutes for glutamic acid, 45 minutes forkainic acid and 10 minutes for NMDA exposure, after which time the mediawas replaced with 100 μl of 50% Neurobasal/2% N2 supplement (Invitrogen)and 50% balanced salt solution (BSS; see below). Cultures were incubatedfor a further 24 hours at 37° C. in the CO₂ incubator. The untreatedcontrols with or without glutamic acid or kainic acid treatment receivedthe same wash steps and media additions.

In one experiment, following the 15 minute CPP incubation (5 or 10 μM),the media in wells was removed and wells washed once in 300 μl of BSSbefore the addition of Neurobasal/2% B27 containing glutamic acid (100μM/100 μl). Following this step, cultures were treated as describedabove. Untreated controls with or without glutamic acid exposurereceived the same wash steps and media additions. In addition, apost-glutamic acid exposure CPP treatment (5 μM) experiment wasperformed for the R9 peptide and the JNK1-1D-TAT control peptide. Inthis experiment, neurons were exposed to glutamic acid (100 μM) in 100μl Neurobasal/2% B27 for 5 minutes as described above, after which timethe media was removed and replaced with 50 μl Neurobasal/2% N2supplement, followed by peptide (10 μM/50 μl in BSS) addition at 0 and15 minutes post-glutamic acid exposure.

For pre-glutamic acid exposure experiments, neurons were exposed topeptide(s) for a 10 minute period, immediately before or 1, 2, 3, 4 or 5hours prior to glutamic acid exposure. This was performed by removingmedia and adding 50 μl of Neurobasal/2% B27 containing peptide. Afterthe 10 minutes at 37° C. in the CO₂ incubator, media was removed andreplaced with 100 μl of Neurobasal/2% B27 (for immediate glutamic acidexposure media contained glutamic acid; 100 μM). At the relevant peptidepre-treatment time, media was removed and replaced with 100 pi ofNeurobasal/2% B27 containing glutamic acid (100 μM). Following 5-minuteglutamic acid exposure, neuronal culture wells were treated as describedabove. For all experiments untreated controls with or without glutamicacid treatment underwent the same incubation steps and media additions.

Heparin Experiments

Heparin (for injection) was obtained from Pfizer (1000 IU/ml). Twodifferent heparin experiments were performed: 1. Peptides were incubatedwith heparin (20 IU/ml) in Neurobasal/B27 for 5 minutes at, roomtemperature, before addition to culture wells (50 μl) for 15 minutes at37° C. in the CO₂ incubator. Following the incubation period, media inwells was removed and replaced with 100 μl of Neurobasal/2% B27containing glutamic acid (100 μM), and subsequently treated as describedabove; 2. Media in wells was replaced with Neurobasal/2% B27 containingheparin (50 μl; 40 IU/ml) and incubated for 5 minutes at 37° C. in theCO₂ incubator. After the incubation period, peptides or glutamatereceptor blockers (MK801/CNQX) in Neurobasal/B27 (50 μl) were added tothe culture wells and cultures incubated for a further 10 minutes at 37°C. in the CO₂ incubator. Following the incubation period, media in wellswas removed and replaced with 100 μl of Neurobasal/2% B27 containingglutamic acid (100 μM), and subsequently treated as described above. Forall experiments, non-heparin treated peptide controls with glutamic acidtreatment underwent the same incubation steps and media additions.

In Vitro Ischemia/OGD Model and Peptide Incubation

The in vitro ischemia model used for primary cortical neuronal cultureswas performed as previously described (Meloni et al. 2011), Briefly,culture media was removed from wells (glass 96-well plate format) andwashed with 315 μl of glucose free balanced salt solution (BSS; mM: 116NaCl, 5.4 KCl, 1.8 CaCl₂, 0.8 MgSO₄, 1 NaH₂PO₄; pH 6.9) before theaddition of 60 μl BSS containing cell penetrating or control peptides(see Table 1). A non-peptide positive control consisting of theglutamate receptor blockers (5 μM MK801/5 μM 6-cyano-7-nitroquinoxaline:MK801/CNQX) was also included. In vitro ischemia was initiated byplacing wells in an anaerobic incubator (Don Whitely Scientific,England; atmosphere of 5% CO₂, 10% H₂ and 65% argon, 98% humidity) at37° C. for 55 minutes. Upon removal from the anaerobic incubator, 6.0 μlof Neurobasal/2% N2 supplement was added to the wells and culturesincubated for a further 24 hours at 37° C. in the CO₂ incubator. Controlcultures received the same BSS wash procedures and media additions asischemic treated cultures before incubation at 37° C. in the CO₂incubator.

For pre-OGD exposure experiments the procedure was the same as describedin the glutamic acid model, except the 1.0 minute peptide pre-treatmentwas performed using 100 μl Neurobasal/2% B27. Control cultures underwentthe same BSS wash procedures and media additions as OGD treatedcultures.

The in vitro ischemia model was also used for bEND3 cells, SH-SY5Ycells, and astrocytes. For bEND3 cells anaerobic incubation was extendedto 2-3 hours and upon removal from the anaerobic incubator, 60 μl ofDMEM/2% FCS was added to wells. For SH-SY5Y cells the first BSS washstep (315 μl) was omitted and anaerobic incubation was extended to 2-5hours. Upon removal from the anaerobic incubator, 60 μl of DMEM/2% FCSwas added to wells. For astrocytes, anaerobic incubation was extended to1:15 to 2:00 hours and upon removal from the anaerobic incubator, 60 μlof DMEM/2% FCS was added to wells.

In Vitro Neuronal, bEND3 and Astrocyte Toxicity Model and PeptideIncubation

For neuronal cultures peptides were added to culture wells (96-wellplate format) by removing media and adding 100 μl of 50% Neurobasal/2%N2 supplement and 50% BSS containing CPPs, JNKI-1D-TAT or TAT-NR2B9c.Control cultures received of 50% Neurobasal/2% N2 supplement and 50% BSSmedia only. Cultures were incubation at 37° . in the CO₂ incubator for20 hours, after which time cell viability was assessed using the MTSassay. For bEND3 cultures, peptides were added to culture wells (96-wellplate format) by removing media and adding 100 μl of DMEM/2% FCScontaining peptide. Control cultures received 100 μl of DMEM/2% FCSonly. Cultures were incubation at 37° C. in the CO₂ incubator for 0.5, 1or 2 hours, after which time cell viability was assessed using the MTSassay. For astrocyte cultures peptides were added to culture wells(96-well plate format) by removing media and adding 100 μl of DMEM/2%FCS containing peptide. Control cultures received 100 μl of DMEM/2% FCSonly. Cultures were incubation at 37° C. in the CO₂ incubator for 24hours, after which time cell viability was assessed using the MTS assay.

Cell Viability Assessment and Statistical Analysis

Twenty-four hours after insult, neuronal cultures were examined by lightmicroscopy for qualitative assessment of neuronal cell viability.Neuronal viability was quantitatively measured by3-(4,5,dimethyliazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazoliumsalt (MTS) assay (Promega, Australia), The MTS assay measures thecellular conversion of the tetrazolium salt to a water-soluble brownformazan salt, which is detected spectrophotometrically at 490nm. MTSabsorbance data were converted to reflect proportional cell viabilityrelative to both the untreated and treated controls, with the untreatedcontrol taken as 100% viability, and presented as mean±SEM. For studiesusing astrocytes, bEND3, and SH-Y5Y cells, raw MTS data was used togenerate graphs. Viability data was analysed by ANOVA, followed bypost-hoc Fischer's PLSD test, with P<0.05 values consideredstatistically significant. Four- to six wells were used in all assays.

Rat Permanent Focal Cerebral Ischemia Model—Experimental groups andtreatments

All treatments were randomized and administered in a blinded manner,Administration of peptide (DR9: rrrrrrrrr-NH2; R12, R15, R18, protaminesulphate; protamine) or vehicle (saline: 0.9% NaCl) treatment solutionwas performed at 30 min post-MCAO. The peptide treatment comprised DR9in saline (600 μl) to provide an intravenous loading dose of 1 μmol/kgor 1000 nmol/kg given over 5-6 min via the right jugular vein.

Rat Permanent Focal Cerebral Ischemia Model

This study was approved by the Animal Ethics Committee of the Universityof Western Australia. Male Sprague Dawley rats weighing 270 g to 350 gwere kept under controlled housing conditions with 12 hour light-darkcycle with free access to food and water. Experimental animals werefasted overnight and subjected to permanent middle cerebral arteryocclusion (MCAO) as follows.

Anaesthesia was induced with 4% isoflurane and a 2:1 mix of N₂O and O₂via mask. Anaesthesia was maintained at 1.7-2% isoflurane. Cerebralblood flow (CBF) was monitored continuously using laser Dopplerflowmetry (Blood FlowMeter, AD Instruments, Sydney, Australia). Theprobe was located 1 mm caudal and 4 mm lateral (right) to the bregma. Acannula was inserted in the right femoral artery to continuously monitorblood pressure and to provide samples for blood glucose and blood gasreadings. Blood glucose was measured using a glucometer (MediSenseProducts, Abbott Laboratories, Bedford, Mass., USA) and blood gases weremeasured using a blood gas analyser (ABL5, Radiometer, Copenhagen,Denmark). Blood pressure was maintained at 80-100 mmHg. During surgery,rectal temperature was maintained at 37±0.5° C. and warming applied witha fan heater when necessary. For intravenous infusions, a length of PVCline primed with heparinised saline was tied in place in the rightjugular vein, then externalised through a dorsal mid-scapular incisionto a tether/swivel system (Instech Laboratories, Philadelphia, USA)designed to permit free movement.

The right common carotid artery (CCA) was exposed via a ventral neckincision. The external carotid artery (ECA) was isolated aftercauterisation of the superior thyroid and occipital arteries. Theisolated section of the ECA was ligated and cauterised to create astump. The carotid body was removed and the pterygopalatine artery wasligated. A 4-0 nylon monofilament with a 0.39 mm diameter silicone tip(Doccol, Redlands, Calif., USA) was inserted through the ECA stump intothe CCA and advanced rostrally into the internal carotid artery (ICA)until the laser Doppler flowmetry recorded a >30% decrease from baselineof cerebral blood flow. The monofilament was secured in two places (atthe base of the ECA stump and on the)CA) for the remainder of theexperiment. Animals were given post-operative analgesia consisting ofpethidine mg/kg intramuscular) and bupivacaine (1.5 mg/kgsubcutaneously) at head and leg incision sites.

Post-surgery animals were allowed to recover in a climate-controlledchamber and their core body temperature monitored and maintained at37±0.5° C. by a cooling/heating fan when required for 3-4 hours.

Tissue processing and infarct volume measurement

Animals were sacrificed 24 hours post-MCAO with intra-peritonealinjections of sodium pentobarbitone (900 mg/kg). After euthanasia, thebrain was removed and placed in a sterile container of 0.9% NaCl andthen placed in a −80° C. freezer for 7 minutes. The brain was thencoronally sliced from the junction of the cerebellum and cerebrum to 12mm rostral to this point in 2 mm thick slices. Slices were immediatelystained with 1% 2,3,5 triphenyltetrazolium chloride (TTC, Sigma, StLouis, Mo., USA) at 37° C. for 20 minutes, followed by fixation in 4%formalin at room temperature for at least 18-24 hours before infarctvolume measurement. Slices were scanned and images were analysed by anoperator blind to treatment status using ImageJ 3^(rd) edition (NIH,USA). The total infarct volume was determined by adding the areas ofinfarcted tissue on both sides of the 2 mm sections, These measuredareas were multiplied by half slice thickness (1 mm), and corrected forcerebral oedema by multiplying the ratio of affected to normalhemisphere areas.

Statistical Analysis

For infarct volume measurements, the peptide treatment group wascompared to the vehicle control group by student West (R90 trial) orANOVA, followed by post-hoc Fischer's PLSD test (R12, R15, R18 and Ptmtrial).

Experimental Example 1 Neuroprotection Following Glutamic Acid Exposure

The CPPs TAT-D, R9, and penetratin provided significant neuroprotectionin a dose response manner (FIG. 1a , Table 3), Visual assessment ofcultures post-injury also confirmed the neuroprotective effect thatranged from ≈5% for untreated glutamic acid exposed cultures to 100%survival for R9 treated cultures. R9 was the most potent peptide with anIC50 value of 0.78 μM, followed by penetratin (IC50: 3.4 μM) and TAT-D(IC50: 13.9 μM). The Pep-1 peptide was ineffective. The glutamatereceptors blockers and control peptides (JNKI-1D-TAT, PYC36L-TAT) werealso highly effective in this model (FIG. 1a ).

In addition, the TAT-D peptide displayed a similar level ofneuroprotection as the TAT-L peptide (FIG. 1b ). When CPPs werewashed-out prior to glutamic acid exposure only R9 (in this experiment)displayed high level neuroprotection (FIG. 1c ).

The R9 and R12 peptides were also highly effective when addedimmediately after glutamic acid exposure, and R9 mildly effective whenadded at 15 minutes post-insult. In contrast, the JNKI-1D-TAT and NR29c(also referred to as TAT-NR2B9c) peptides did not significantly increaseneuronal survival when added immediately after, or at 15 minutespost-glutamate exposure (FIG. 1d ).

FIGS. 1e-j provide additional efficacy data in the glutamate model forpeptides R1, R3, R6, R9, R12, R15, R18, R9/E9 (also referred to asE9/R9), R9/tPA/R9 (also referred to as R9/X7/R9) as well as controlpeptides JNKI-1D-TAT and NR29c.

In dose response studies using R1, R3, R6, R7, R8, R9, R12, R15 and R18in the glutamate model revealed that: i) R1, R3, R6 and R7 displayed noto little neuroprotection; ii) R8 displayed neuroprotection at 5 μM,(iii) the order of potency for the other peptides was R15>R18>R12>R9;and iv) neuroprotective efficacy for R15 and R18 was reduced at thehigher concentration (5 μM); see Table 4, FIGS. 1 e, 1 l, 1 j, 6, 7, and8.

In a dose response study using R9, DAHK (last four end N-terminal aminoacids of protein albumin) PTD4 (modified TAT peptide with 33× increasedcell penetrating ability; (Ho et al, 2001), R9/E9 (Arg-9/Glu-9; neutralpeptide) and R9/tPA/R9 (tissue plasminogen activator enzyme peptidecleavage site flanked by R9) in the glutamate model revealed that: I)PTD4 and R9/E9 displayed no to little neuroprotection; DAHK had lowlevel neuroprotection; and ii) R9/tPA/R9 had a potency between R12 andR18. Also the TAT-NR29C peptide (C-terminal NR2B NMDRA receptor subunitpeptide; blocks NMDAR signaling with PSD-95 protein to block NO (nitrousoxide) production; Aarts et al 2002) was ineffective. See Table 4 andFIGS. 1 d, g, h.

Peptide R12 was more effective than R9 (based on neuroprotection at0.625 μM concentration) and both were more effective compared toPCY-36-TAT, in the glutamic acid model, while NR29c was ineffective(FIG. 1g ). Peptide R12 was more effective than R9 (based onneuroprotection at 0.5 μM and 1 μM concentrations, while R1, R3, R6 andNR29c were ineffective (FIG. 1i ). Peptide R9 synthesized by twodifferent companies also had similar efficacy in the glutamate model(FIG. 1j ).

Peptides R9, R12, R15 and R18 were effective when added to neuronalcultures during the 5 minute glutamic acid insult only (FIG. 28), orremoved from cultures prior to the insult following a 10 minutepre-exposure (FIG. 29).

Peptides R12 and R15 were effective when added to neuronal cultures for10 minutes, 1 to 4 hours prior to the glutamic acid insult (FIG. 30).

Peptides PTD4 shows low level neuroprotection, and peptide E9/R9 showsno protection in the glutamic acid model (FIG. 33), while arginine-richpeptides XIP and NCXBP3 show high levels of protection (FIGS. 34, 35).Poly-lysine-10 peptide (K10) and TAT-NR2B9c peptides show low levelneuroprotection in glutamic acid (FIG. 36) and NMDA models (FIG. 37),respectively. R8 and R9 fused to calpain cleavage site (Cal/R9) showmoderate to high level neuroprotection in glutamic acid (FIG. 38). FIGS.39 and 40 show that the negatively charged molecule heparin blocksneuroprotective actions of R9D, R12, R15, and PYC36-TAT, but notglutamate receptors blockers (5 μM K801/5μM CNQX) in the glutamic acidmodel. FIG. 41 shows that the JNKI-1 peptide when fused to the(non-arginine) kFGF CPP, which does not rely on endocytosis for uptake,was not neuroprotective in the glutamic acid model; the kFGF peptide wasalso ineffective. In contrast, TAT-JNKI-1 and JNKI-1-TATD wereneuroprotective. FIG. 44 shows that peptides R9D, R12, R15, andPYC36-TAT, TAT, TAT-NR2B9c, TAT-JNKI-1 and JNKI-1-TATD and glutamatereceptors blockers (5 μM K801/5 μM CNQX) to varying degrees reducedcalcium influx in neuronal cultures after treatment with glutamic acid.

Experimental Example 2

Neuroprotection Following Kainic acid Exposure

Following kainic acid exposure TAT-D, R9 and penetratin wereneuroprotective, but less effective than in the glutamic acid model, anddid not always display a typical dose response pattern (FIG. 2, Table2). Pep-1 was ineffective. R9 was the most potent peptide, increasingneuronal survival from 20% to a maximum of 80%. The respective IC50values for R9, penetratin, and TAT-D were 0.81, 2.0 and 6.2 μM. Theglutamate receptors blockers, JNKI-1D-TAT and PYC36L-TAT were alsoeffective in this model (FIG. 2).

Experimental Example 3 Neuroprotection Following in VitroIschemia/Oxygen Glucose Deprivation (OGD)

Following in vitro ischemia all four CPPs displayed neuroprotectiveeffects (FIG. 3, Table 2). Neuroprotection with Arg-9 (IC50: 6.0 μM) andTAT-D (IC50: 7.1 μM) was similar; efficacy followed a dose responsepattern and increased neuronal survival from ≈10% to 40-50%.Neuroprotective efficacy was lost with increasing concentrations ofpenetratin (a5 μM), while Pep-1 was only neuroprotective at lowerconcentrations (1-5 μM). Glutamate receptors blockers and PYC36L-TATwere also effective in this model (FIG. 3a ).

In addition, when added post-in vitro ischemia R9, R12, R15 and R18displayed neuroprotective effects, however higher concentrations of R15and R18 reduced efficacy (FIG. 3b ).

R9 also reduced bEND3 and SH-5YSY cell death when exposed to in vitroischemia (FIG. 3c ). RIB also reduced astrocyte cell death when exposedto in vitro ischemia (FIG. 42).

FIG. 6 shows a dose response study using R9, R10, R11, R12, R13 and R14in the glutamate model, which revealed that all peptides displayedsignificant neuroprotection between 1 and 5 μM, except for R11 whichdisplayed significant neuroprotection at 2 and 5 μM. Mean±SEM: N =4; *P<0.05. (Peptide concentration in μM).

FIG. 7 shows a dose response study using R9D, R13, R14 and R15 in theglutamate model, which revealed that all peptides displayedneuroprotection between 1 and 5 μM. Mean±SEM: N =4; * P<0.05. (Peptideconcentration in μM).

As shown in FIG. 8, in a dose response study using R6, R7, R8, and R9 inthe glutamate model, it was revealed that peptides R8 and R9 displayedsignificant neuroprotection at 5 μM and 1 μM and 5 _(μM) concentrations,respectively, while R6 and R7 did not exhibit significantneuroprotection. Mean±SEM: N=4; * P<0,05. (Peptide concentration in μM).FIG. 31 shows a dose response study using R9, R12, R15 and R18 whenpeptides were added for 15 minutes after OGD. Neuroprotection isdisplayed for R12, R15 and R18, but not R9 at 1 μM and 5 μM.

Peptides R12 and R18 were effective when added to neuronal cultures for10 minutes, 1 to 3 hours prior to OGD (FIG. 32).

TABLE 3 IC50 values of cell penetrating and control peptides for thethree injury models IC50: IC50: IC50: Glutamic Kainic In vitro SEQ acidmodel acid model ischemia model Peptide ID NO. (μM) (μM) (μM) Arg-9 (R9)6 0.78  0.81 6.0 TAT-D 20 13.9 8.2 7.1 Penetratin 22 3.4 2.0 N/A Pep-123 N/A N/A >15 PYC36L-TAT# 24 1.5 — — JNKI-1D-TAT# 25 2.1 6.5 — * Basedon dose response graphs shown in FIG. 1a, FIG. 2 and FIG. 3a. #IC50values for JNKI-1D-TAT and PYC36L-TAT peptides from Meade et al. (2010a,b). N/A = not applicable because peptides were either ineffective orincreased cell death at higher doses. — = data not available.

TABLE 4 IC50 values of polyarginine peptides for glutamic acid models*IC50: Glutamic 95% confidence Peptide SEQ ID NO. acid model (μM)intervals (μM) Arg-1 (R1) 1 >5 μM N/A Arg-3 (R3) 2 >5 μM N/A Arg-6 (R6)3 >5 μM N/A Arg-9 (R9) 6 0.83 0.16-4.1 Arg-12 (R12) 9 0.44 0.16-1.2Arg-15 (R15) 12 0.19 0.06-0.6 Arg-18 (R18) 13 0.24  0.08-0.75 R9/tPA/R916 0.29 0.06-1.4 *Based on dose response graph shown in FIG. 1e. N/A =not applicable because peptides had no or little effect at highestconcentration tested (5 μM).

Experimental Example 4 Animal Trial

In an initial animal trial that was conducted, it was shown that Arg-9(R9), R18 and protamine (Ptm) possessed neuroprotective activity inviva. These trials showed the efficacy of R9D peptide in rat permanentmiddle cerebral artery occlusion (MCAO) stroke model. R9D peptide wasadministered intravenously 30 min post-MCAO. Infarct volume (braininjury) was measured 24 h post-MCAO (mean±SEM). This is shown in FIGS. 5and 27 where it can be seen that (Ns=8-12 animals for each group)treatment with R9D, R18 and protamine (Ptm) showed a statisticallysignificant neuroprotective effect by reducing infarct volume (braindamage) by approximately 20% after a MCAO stroke.

General Observations and Discussion

The Applicant assessed TAT as known neuroprotective example and threeother CPPs (penetratin, R9, and Pep-1) for their neuroprotectiveproperties in cortical neuronal cultures following exposure to glutamicacid, kainic acid, or in vitro ischemia (oxygen-glucose deprivation).

In addition, polyarginine peptides (R9, R12, R15, R18) and/orarginine-rich protamine peptide were also assessed in astrocyte, brainendothelial cell line (bEND3), and/or a neuroblastoma cell line(SH-SY5Y) cultures using the in vitro ischemia model.

R9, penetratin and TAT-D displayed consistent and high levelneuroprotective activity in both the glutamic acid (IC50: 0.78, 3.4,13.9 μM) and kainic acid (IC50; 0.81, 2.0, 6.2 μM) injury models, whilePep-1 was ineffective.

The TAT-D isoform displayed similar efficacy to the TAT-L isoform in theglutamic acid model. R9 displayed efficacy when washed-out prior toglutamic acid exposure. However, R9 was significantly more effectivethan peptides that had previously been shown to be neuroprotective, i.e.TAT-D, TAT-L, PYC36L-TAT, and JNKI-1D-TAT.

Neuroprotection following in vitro ischemia was more variable, with allpeptides providing some level of neuroprotection (1050; R9: 6.0 μM,TAT-D: 7.1 μM, penetratin/Pep-1; >10 μM). The positive control peptidesJNKI-ID-TAT (JNK inhibitory peptide) and/or PYC36L-TAT (AP-1 inhibitorypeptide) were neuroprotective in all models.

In a post-glutamic acid treatment experiment, R9 was highly effectivewhen added immediately after, and mildly effective when added 15 minutespost-insult, while the JNKI-1D-TAT control peptide was ineffective whenadded post-insult.

In an initial animal trial that was conducted, it was shown that R9,R12, R18, and protamine possessed neuroprotective activity in viva.

In a dose response study using R1, R3, R6, R9, R12, R15 and R18 in theglutamate model revealed that: i) R1, R3, R6, and R7 displayed no tolittle neuroprotection; ii) the order of potency for the other peptideswas R15 >R18>R12>R9; and iii) neuroprotective efficacy for R15 and R18was reduced at the higher concentration tested (5 μM).

In a dose response study using R9, DAHK (last four end N-terminal aminoacids of protein albumin), PTD4 (modified TAT peptide with X33 increasedcell penetrating ability; Ho et al, 2001), R9E9 (R9/Glu-9; neutralpeptide) and R9/tPA/R9 (tissue plasminogen activator enzyme peptidecleavage site flanked by R9) in the glutamate model revealed that: i)PTD4 and R9/E9 displayed no to little neuroprotection; DAHK had lowlevel neuroprotection; and ii) R9/tPA/R9 had a potency between R12 andRIB. Also the TAT-NR2B9C peptide (C-terminal NR2B NMDRA receptor subunitpeptide blocks NMDAR signaling with PSD-95 protein to block. NOproduction; Aarts et al., 2002). Polyarginine and arginine-rich(protamine) peptides could also reduce astrocyte, bEND3, and SH-SY5Ycell death following in vitro ischemia.

These findings demonstrate that the peptides of the invention have theability to inhibit neurodamaging events/pathways associated withexcitotoxic and ischemic injuries. Poly-arginine peptides with 9arginine amino acid residues are particularly neuroprotective.

The cytoprotective properties of the peptides of the invention suggeststhey are ideal carrier molecules to deliver neuroprotective drugs to theCNS following injury and/or to serve as potential neuroprotectants intheir own right.

The peptides of the invention thus exhibit neuroprotective properties indifferent in vitro injury models that have been shown to be translatableinto in vivo models. This is further bolstered by the neuroprotectiveeffect shown in the initial animal trials that were conducted. Thesuperior neuroprotective action of R9 was surprising; based on 1050values R9 was 17 and 7 fold more potent than TAT-D in glutamic acid andkainic acid models respectively, and was the only peptide effective evenwhen washed-out prior to glutamate acid exposure. This finding suggeststhe increased arginine residues and/or the slightly higher net charge(10 vs 9 at pH 7) of R9 are important factors for neuroprotectionfollowing excitotoxicity. Furthermore, while the exact reason for theloss of efficacy of TAT and penetratin, but not R9 following wash-outprior to glutamic acid exposure is unclear, it may relate to the speedof R9 intracellular up-take, rather than an extracellular mechanism.This is supported by the finding that R9 was effective when added afterglutamic acid exposure, while the JNKI-1 D-TAT peptide was ineffective.

Furthermore, when arginine and polyarginine peptide(s) R1, R3, R6, R9,R12, R15 and R16 were assessed in the glutamic acid injury model onlypeptides R9, R12, R15 and R18 showed significant neuroprotection at thedoses tested; R15 appeared to be the most potent peptide. A hybrid R9peptide (R9/tPA/R9) containing the tPA cleavage linker site was alsohighly effective in the glutamic acid model. R9 was also more effectivethan R12 when added post-glutamic acid exposure. Interestingly, PTA4(modified TAT peptide with 33× improved transduction efficacy whencompared to regular TAT; Ho et al, 2001), the R9/E9 hybrid and the NR29c(a TAT fused peptide that blocks NMDA/glutamate receptor-induced NOproduction) and DAHK (last four end N-terminal amino acids of proteinalbumin) were largely ineffective following glutamic acid exposure.

Peptides R9, R12, R15 and R18 were also effective in the in vitroischemia model when added after anaerobic incubation (i.e. duringreperfusion phase of injury). R9 was also able to protect brainendothelial cells (bEND3 cells) and neuroblastoma cells (SH-5YSY cells)following in vitro ischemia.

As mentioned hereinbefore, a result in the study was the demonstrationthat penetratin and Pep-1 also exhibited neuroprotective properties. Thepenetratin and Pep-1 peptides bear no amino-acid sequence relatedness toeach other, or the TAT/R9 peptides. Interestingly penetratin was highlyneuroprotective in the excitotoxic models (IC50s: 3.4 and 2 μM), butless effective in the in vitro ischemia model, with increasingconcentrations reducing efficacy. The Pep-1 peptide was generallyineffective in the excitotoxic models and in some experiments appearedto increase neuronal death (data not shown), but was neuroprotectivefollowing in vitro ischemia at lower concentrations. Interestingly, whenpenetratin was washed-out from neuronal cultures prior to glutamic acidexposure, visual observations revealed that the peptide did display someearly neuroprotective effects (data not shown). Hence, both penetratinand Pep-1 behaved differently to each other and the TAT/R9 peptides inthe injury models.

The differential neuroprotective responses for the four CPPs in theexcitotoxic and ischemic injury models is likely to be related to thepeptides' physical-chemical properties, and more specifically theirendocytic-inducing properties. Furthermore, it is likely that theneuroprotective action of the CPPs is mediated at the cell membrane(e.g. receptors, ion channels). Xu et al (2008), have suggested that TATmay alter the cell membrane and thereby affect the function of cellsurface receptors, such as the NMDA receptor, resulting in reducedcalcium influx.

It is contemplated, however, that the peptide or peptides of theinvention can act to block NMDA receptor functioning and/or block,downregulate, or decelerate the influx of calcium. An alternativemechanism is that the CPPs interact and stabilise the outermitochondrial membrane and thereby help to preserve mitochondrialfunction. Potential benefits are maintenance of ATP synthesis, reducedreactive oxygen species production, and improved calcium handling. Tothis end, the Applicant has observed that Arg-9 can increase MTSabsorbance levels above baseline levels in normal neurons and followinginjury (e.g. FIG. 1A, 15 μM). Since reduction of MTS to its formazanproduct primarily occurs in mitochondria, the ability of Arg-9 toincrease formazan levels is supportive that the peptide is improvingmitochondrial function. Another potential mechanism especially inrelation to R9 and TAT, is that these arginine rich peptides areinhibiting the calcium-dependent pro-protein convertase enzyme furin(Kacprzak et al. 2004), and thereby blocking activation of potentiallydamaging proteins.

The invention demonstrates that cationic amino acid rich CPP,particularly arginine-rich CPPs or carrier-peptides (e.g. R12, R15,protamine), display a high level of neuroprotection, as opposed to CPPsin general. This raises the possibility that the mechanism of action ofa neuroprotective peptide fused to a CPP is largely, if not exclusivelythe result of an enhanced neuroprotective effect of the carrier-peptide.Furthermore, the mechanism by which arginine-rich CPPs exert theirneuroprotective action may be linked to endocytosis, a predominantcarrier-peptide cellular uptake route, rather than by an interactionwith a specific cytoplasmic target. In contrast, a neuroprotectivepeptide fused to a carrier-peptide entering a cell by endocytosis mustfirst escape the endosome, which is known to be a highly inefficientprocess (Al-Taei et al., 2006; El-Sayed et al., 2009; Appelbaum et al.,2012; Qian et al., 2014), before it can interact with its cytoplasmictarget, hereby rendering it highly unlikely that the peptide can actthrough interaction with its intended target.

With respect to CPP intracellular entry, the predominant mechanism isconsidered to be by endocytosis (macropinocytosis) (Palm-Apergi et al.2012). Although less relevant to the present invention, a recent reporthas demonstrated that cargo properties may also promote a direct cellentry mechanism by certain CPPs (Hirose et al. 2012). However, what ispotentially highly relevant is how specific cargos, peptide orotherwise, may affect CPPs by enhancing their neuroprotective action,improving translocation efficiency and/or as demonstrated by Cardozo etal (2007) increasing their toxicity. This is especially important whenthe cargo itself is neuroprotective, because as mentioned above, thismakes discerning the neuroprotective effect between the CPP and thecargo very difficult. For example, in a previous studies (Meade et al.2010a), the addition of three amino acid residues (Pro, Lys, Ile) fromthe PYC36 peptide to the TAT-D peptide (AMBD-TAT) resulted in 1050values decreasing from >15 μM for TAT-D to 1.1 μM for AM8D-TAT in theglutamic acid model.

A positive effect with the TAT peptide control has not always beenobserved. There are a number of possible explanations and to addressthis question: it is first necessary to differentiate studies using theTAT peptide only (i.e. GRKKRRQRRRG), versus studies using TAT fused to areporter protein (e.g. GFP, β-gal) or peptide (e.g. HA and/or 6× HIStag, scrambled peptide) as a control. With respect to the studies thathave used the TAT peptide by itself as a control, it is possible TAT wasineffective at the dose used and/or the injury model was too severe touncover a neuroprotective effect. For example, Boresello et al. (2003)did not detect a neuroprotective effect with the TAT peptide following a12, 24 or 48 hour exposure of cortical neuronal cultures to 100 μM NMDA.In contrast the L-JNKI-1 peptide was effective at 12 and 24 hours, whilethe protease resistant D-JNKI-1 peptide was effective at alltime-points. Given the superior efficacy of the JNK1-1 peptides comparedto the TAT peptide, it is possible that at the concentration tested, TATwas not neuroprotective or that any neuroprotective effects wereoverridden due to NMDA insult severity. In a study by Ashpole and Hudmon(2011) a modest protective effect with the TAT peptide was observed incortical neuronal cultures following glutamic acid exposure.Furthermore, the authors concluded that since the TAT peptide providedlittle protection, the neuroprotection observed for their CAMKIIinhibitory peptide was not due to the “import sequence” (i.e. TAT).However, it cannot be ruled out that the CAMKII inhibitory peptideincreased the potency of the TAT peptide. Lastly, it is possible thatthe TAT peptide is only neuroprotective in specific injury models andcell types.

In studies using TAT fused to a reporter protein or control peptide, inaddition to the points raised above, it is also likely that the controlprotein/peptide may act to dampen or nullify the TAT peptide'sneuroprotective properties. Based on the many studies that have usedTAT-fused proteins/peptides as controls and showed no neuroprotectiveeffects, this would appear to be the case (e.g. Kilic et al. 2003;Doeppner et al. 2009). It needs to be borne in mind, however, that themere fact that a protein is a CPP does not necessarily mean that it willbe neuroprotective, which is borne out by the fact that PTD-4 peptide (amodified TAT peptide with 33× better transduction efficiency than TATitself; Ho et al, 2001) has little to no neuroprotective properties.

TABLE 5 Amino acid sequences of different protamine (salmon) peptides;amino acid residues/arginine residues and molecular weights. SEQ Name ofOther aa′s/arg MW ID NO.: Sequence Type information Sequence residues(da) — Protamine Poly- Injectable/I Protamine 1-Protamine 4¹ 32/21≈4,500 sulphate peptide V form mixture; mixture Ptm 32 Protamine 1;Poly- Peak 1 PRRRRRSSSRPIRRRRRPRASRRRRRGG 32/21  4,236 Ptm1 peptideHPLC^(a) RRRR 33 Protamine 2; Poly- Peak 2 PRRRRSSRRPVRRRRRPRVSRRRRRGGR31/21  4,163 Ptm2 peptide HPLC^(a) RRR 34 Protamine 3; Poly- Peak 3PRRRRSSSRPVRRRRRPRVSRRRRRGGR 31/20  4,094 Ptm3 peptide HPLC^(a) RRR 35Protamine 4; Poly- Peak 4 PRRRRASRRIRRRRRPRVSRRRRRGGRR 30/21  4,064 Ptm4peptide HPLC^(a) RR 36 Protamine 5; Poly- SwissProt^(c)PRRRRSSSRPVRRRRRPRVSRRRRRRGG 32/21  4,250 Ptm5 pepoide RRRR 37Low molecular Poly- Derived VSRRRRRRGGRRRR 14/10  1,880 weight pepoidefrom protamine protamine^(b) (LMWP) 38 Protamine 5 Ptm 5, SwissProt^(c)5′ATGCCCAGAAGACGCAGATCCTCCAG N/A N/A nucleotide DNACCGACCTGTCCGCAGGCGCCGCCGCCCT sequence codingAGGGTGTCCCGACGTCGTCGCAGGAGAG sequence GAGGCCGCAGGAGGCGT-3′ (Protaminipeptide sequences (salmon) present in protamine sulphate for clinicaluse or SwissProt data base. [a = Peaks 1-4 identified following HPLC ofprotamine sulphate^(1;) b = Sequence in SwissProt (Swissprot: P14402).(1) Hoffmann JA1, Chance R E, Johnson M G. 1990. Purification andanalysis of the major components of chum salmon protamine contained ininsulin formulations using high-performance liquid chromatography.Protein Expr Purif. 1(2): 127-33. (2) Chang L C, Lee H F, Yang Z, Yang VC. 2001. Low molecular weight protamine (LMWP) as nontoxic heparin/lowmolecular weight heparin antidote (I): preparation and characterization.PharmSci. 3(3): E17).

Experimental Example 1 Neuro Protection Following Glutamic Acid Exposure

Protamine sulphate (protamine; Ptm) provided significant neuroprotectionin a dose response manner (FIGS. 9, 10, 12, 13, 15). Visual assessmentof cultures post-injury also confirmed the neuroprotective effect thatranged from 5% for untreated glutamic acid exposed cultures to 85-100%survival for protamine treated cultures. In addition, a 5 or 10 minuteprotamine pre-exposure was also highly neuroprotective resulting 100%neuronal survival (FIG. 11). In dose response experiments, low molecularweight (LMWP), protamine 1 (Ptm1), protamine 2 (Ptm2), protamine 3(Ptm3), protamine 4 (Ptm4), protamine 5 (Ptm5), peptide were alsoneuroprotective (FIGS. 12, 13, 15).

In protamine and LMWP pre-exposure experiments, protamine wasneuroprotective when neurons where exposed to protamine immediatelybefore and 1 or 2 hours prior to glutamic acid insult, while LMWP wasonly neuroprotective when exposure was immediately before glutamic acidinsult (FIG. 14).

Experimental Example 2 Neuroprotection Following Oxygen-glucoseDeprivation (OGD)

In the OGD model protamine was neuroprotective when neurons were treatedwith peptide 1 hour before insult (FIG. 18) or post-insult (FIGS. 16,17). In addition, when added for 15 or 30 minutes, post-OGD protaminewas also neuroprotective (FIGS. 19, 20, 21, 22), LMWP was notneuroprotective when neurons were pre-exposed to the peptide 1 hourbefore OGD, but it was neuroprotective when added for 15 minutespost-OGD. In addition when protamine peptides (Ptm1-Ptm5) and LMWP wereadded for 15 minutes post-OGD, peptides Ptm2, Ptm4, Ptm5 and LMWP wereneuroprotective (FIG. 22).

Experimental Example 3

Protection of bEND3 cells Following Oxygen-glucose Deprivation (OGD)

In the OGD model, protamine and protamine 4 (ptm4) was protective forblood brain barrier (bEND3) endothelial cells when treated with peptide15 minutes before OGD (FIGS. 23, 24). In addition, exposure of bEND3 toprotamine at concentrations ranging from 1.25 to 15 μM for between 0.5to 2 hours did not cause any significant toxicity based on MTS assay(FIGS. 25, 26). FIG. 43 shows that Ptm1-Ptm5, and low molecular weightprotamine (LMWP) at concentration from 2.5 to 10μM did not causesignificant astrocyte cell death following 24 hour exposure.

General Observations and Discussion

The Applicant assessed the arginine-rich protamine sulphate (protamine;Ptm), protamine peptides 1-5 (Ptm1-Ptm5, SEQ. ID. NOs. 32 to 36,respectively), and low molecular weight protamine (LMWP, SEQ. ID. NO.37) for their neuroprotective properties in cortical neuronal culturesfollowing exposure to glutamic acid or in vitro ischemia (oxygen-glucosedeprivation—OGD). Both injury models are commonly used to mimic theeffects of ischemic stroke.

The Applicant also assessed the use of protamine peptides in protectingblood brain barrier (bEND3) endothelial cells from OGD.

Protamine displayed consistent and high-level neuroprotective activityin both the glutamic acid and OGD injury models, while protamine alsoprovided protection in the bEND3 cells. LMWP was slightly less effective(based on dose concentrations to achieve equivalent neuroprotection asprotamine) in the neuronal glutamic acid and OGD injury models, This ismost likely due to the LMWP peptide containing fewer arginine residues.Protamine peptides 1-5 (Ptm1-Ptm5) were also highly neuroprotective inthe in the glutamic acid model, while Ptm2, Ptm4 and Ptm5 wereneuroprotective in the OGD model.

In a OGD study using bEND3 cells, a 15-minute pre-exposure withprotamine significantly increased cell viability and thus protection,after different duration of OGD (2 h:15 min, 2 h:30 min or 2 h:45 min).In addition, a 15-minute pre-exposure with Ptm4 also significantlyincreased cell viability and thus protection, in the OGD model.

In a bEND3 toxicity study using protamine at varying concentrations, itwas revealed that protamine is not toxic even at concentrations as highas 15

These findings demonstrate that protamine peptides of the invention havethe ability to inhibit neurodamaging events/pathways associated withexcitotoxic and ischemic injuries. Also, due to the effects of protaminein the pre-exposure trials, one new key finding was that protaminetreatment of neurons 1 to 2 hours before glutamic acid or OGD exposurecan induce a neuroprotective response, by reducing cell death. This issignificant because there are a number of cerebrovascular (e.g. carotidendarterectomy) and cardiovascular (e.g. coronary artery bypass graft)surgical procedures where there is a risk a patient can suffer cerebralischemia or a stroke resulting in brain injury. Therefore, protaminepeptides may be able to be given 1-2 hours before such a procedure toprotect the brain against any such cerebral ischaemic event.

The cytoprotective properties of the peptides of the invention suggestthey are ideal neuroprotective drugs for the treatment of CNS injuries.In addition, as they are also likely to have cell penetrating properties[protamine is FDA approved for gene therapy delivery (DNA, viralvectors; Sorgi et al, 1997) and LMWP is used as a cell penetratingpeptide; Park et al, 2005] they are ideal carrier molecules to deliverneuroprotective drugs to the CNS following injury.

The neuroprotective effects of the peptides of the invention are likelyto be related to the peptides' physical-chemical properties.Furthermore, it may well be that the neuroprotective action ofarginine-rich protamine peptides is mediated at the cell membrane (e.g.receptors, ion channels). Studies have suggested that arginine-richpeptides including TAT (Xu et al, 2008), R6 (Ferrer-Montiel et al 1988)and R9-CBD3 (Feldman and Khanna 2013) and TAT-CBD3 (Brustovetsky et al2014) may affect the function of cell surface receptors and ionchannels, such as the NMDA receptor, resulting in reduced calciuminflux. It is contemplated, however, that the peptide or peptides of theinvention can act to block NMDA receptor functioning and/or block,down-regulate, or decelerate the influx of calcium. An alternativemechanism is that the protamine peptides interact and stabilise theouter mitochondrial membrane and thereby help to preserve mitochondrialfunction. Potential benefits are maintenance of ATP synthesis, reducedreactive oxygen species production, and improved calcium handling. Tothis end, the Applicant has observed that protamine can increase MTSabsorbance levels above baseline levels in normal neurons and followinginjury (e.g. FIGS. 11, 12; 5 μM). Since reduction of MTS to its formazanproduct primarily occurs in mitochondria, the ability of protamine toincrease formazan levels is supportive that the peptide is improvingmitochondrial function. Another potential mechanism is that thesearginine-rich peptides are inhibiting the calcium-dependent pro-proteinconvertase enzyme furin (Kacprzak et al. 2004), and thereby blockingactivation of potentially damaging proteins.

With respect to protamine peptide intracellular entry, the predominantmechanism for arginine-rich peptides is considered to be by endocytosis(macropinocytosis) (Palm-Apergi et al. 2012). It is therefore likely,that during peptide endocytosis across the plasma membrane, it resultsin endosomal internalisation of cell surface structures (see FIG. 46).In the setting of neuronal excitotoxicity and ischemia, thedown-regulation of ion channels would be beneficial as it reduces thenormally neuro-damaging influx of calcium and other ions.

The Applicant is of the opinion that they have identified a new class orgroup of peptides that can serve as CPPs as well as neuroprotectivepeptides. The Applicant has found that poly-arginine or arginine-richpeptides, particularly those selected from protamine or Low MolecularWeight Protamine (and mixtures and derivatives thereof, especially inthe form of commercially available protamine sulphate) posses novelneuroprotective or neuro-active properties. There is evidence that thepeptides disclosed herein as part of the invention possess the samerange of neuroprotective or neuro-active properties when used in vivo(Vaslin et al. 2009) and find use in treating neural injuries.

The Applicant has thus found that arginine-rich peptides and CPPsunrelated to TAT posses novel neuroprotective properties, in particularpoly-arginine sequences and sequences of between 10 and 32 amino acidsin length which possess more than 10 arginine residues. (such asprotamine, LMWP, and derivatives thereof). There is evidence that CPPsof the invention possess the same range of neuroprotective propertieswhen used in vivo (Vaslin et al. 2009) and find use in treating neuralinjuries.

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1-30. (canceled)
 31. A method of prophylaxis for neural injury to thebrain of a surgery patient, the method comprising administering to saidsurgery patient a pharmaceutical composition comprising aneuroprotective amount of a poly-arginine peptide having 12 to 32arginine residues, wherein the pharmaceutical composition does notcontain an active pharmaceutical ingredient that is not a poly-argininepeptide.
 32. The method according to claim 31, wherein the patient is acerebrovascular surgery patient.
 33. A method according to claim 32,wherein the patient is a carotid endarterectomy patient.
 34. The methodaccording to claim 31, wherein the patient is a cardiovascular surgerypatient.
 35. The method according to claim 34, wherein thecardiovascular surgery patient is a coronary artery bypass graftpatient.
 36. The method according to claim 31, wherein the poly-argininepeptide has 12 to 18 arginine residues.
 37. The method according toclaim 32, wherein the poly-arginine peptide has 12 to 18 arginineresidues.
 38. The method according to claim 34, wherein thepoly-arginine peptide has 12 to 18 arginine residues.
 39. The methodaccording to claim 31, wherein the method comprises administering thepharmaceutical composition to the patient 0.25 hours to 4 hours prior tothe surgery.
 40. The method according to claim 32, wherein the methodcomprises administering the pharmaceutical composition to the patient0.25 hours to 4 hours prior to the surgery.
 41. The method according toclaim 34, wherein the method comprises administering the pharmaceuticalcomposition to the patient 0.25 hours to 4 hours prior to the surgery.42. The method according to claim 31, wherein the method comprisesadministering the pharmaceutical composition to the patient 0.5 to 3hours prior to the surgery.
 43. The method according to claim 31,wherein the method comprises administering the pharmaceuticalcomposition to the patient 1 to 2 hours prior to the surgery.
 44. Themethod according to claim 31, wherein the method comprises administeringthe pharmaceutical composition within 24 hours of a surgery-associatedneural injury.
 45. The method according to claim 31, wherein the methodcomprises administering the pharmaceutical composition within 5 hours ofa surgery-associated neural injury.
 46. The method according to claim31, wherein the method comprises administering the pharmaceuticalcomposition within 2 hours of a surgery-associated neural injury. 47.The method according to claim 31, wherein the arginine residues of thepoly-arginine peptide consist of D-arginine residues.
 48. The methodaccording to claim 31, wherein the arginine residues of thepoly-arginine peptide consist of L-arginine residues.
 49. The methodaccording to claim 31, wherein the poly-arginine peptide is a mixture ofL-arginine residues and D-arginine residues.
 50. The method of claim 31,wherein said pharmaceutical composition contains an effective amount ofa peptide consisting of 18 arginine residues.
 51. In a surgicalprocedure where the surgery patient is at risk for suffering cerebralischemia or stroke, the improvement that comprises administering to thesurgery patient a pharmaceutical composition comprising an effectiveamount of a poly-arginine peptide having 12 to 32 arginine residues,wherein the pharmaceutical composition does not contain an activepharmaceutical ingredient that is not a poly-arginine peptidepolypeptide.
 52. In the surgical procedure according to claim 51, theimprovement wherein the poly-arginine peptide has 12 to 18 arginineresidues.
 53. A method for promoting the survival of neurons and/orinhibiting neuron death following surgery in a patient scheduled forsurgery, the method comprising administering to said patient apharmaceutical composition comprising an effective amount of apoly-arginine peptide having 12 to 32 arginine residues, wherein thepharmaceutical composition does not contain an active pharmaceuticalingredient that is not a poly-arginine peptide polypeptide, and whereinthe method comprises administering the pharmaceutical composition 0.25hours to 4 hours prior to the surgery.
 54. The method according to claim53, wherein the poly-arginine peptide has 12 to 18 arginine residues.